WEBVTT 00:00:00.500 --> 00:00:02.830 The following content is provided under a Creative 00:00:02.830 --> 00:00:04.370 Commons license. 00:00:04.370 --> 00:00:06.670 Your support will help MIT OpenCourseWare 00:00:06.670 --> 00:00:11.030 continue to offer high-quality educational resources for free. 00:00:11.030 --> 00:00:13.660 To make a donation or view additional materials 00:00:13.660 --> 00:00:17.610 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:17.610 --> 00:00:18.540 at ocw.mit.edu. 00:00:26.045 --> 00:00:27.920 JOANNE STUBBE: OK, so what I want to do today 00:00:27.920 --> 00:00:31.670 is hopefully finish up or get pretty close to finishing up 00:00:31.670 --> 00:00:37.430 module 6, where we've been focused on bacterial uptake 00:00:37.430 --> 00:00:38.855 of iron into cells. 00:00:41.888 --> 00:00:47.720 In the last lecture, I briefly introduced you 00:00:47.720 --> 00:00:51.700 to gram-positive and gram-negative 00:00:51.700 --> 00:00:54.940 big peptidoglycan, small peptidoglycan, 00:00:54.940 --> 00:00:56.690 outer-cell membrane. 00:00:56.690 --> 00:00:58.017 They both have the same goals. 00:00:58.017 --> 00:00:58.850 They've got to get-- 00:00:58.850 --> 00:01:02.900 They take up iron the same way from a siderophore, which 00:01:02.900 --> 00:01:08.365 is what we talked about last time, or by a heme. 00:01:08.365 --> 00:01:09.990 And we'll talk a little bit about that. 00:01:09.990 --> 00:01:12.860 And that's what you focused on in your problem set. 00:01:12.860 --> 00:01:15.170 But they have different apparati to do 00:01:15.170 --> 00:01:19.190 that, because of the differences between the outer-- 00:01:19.190 --> 00:01:21.530 because of the cell walls' distinctions 00:01:21.530 --> 00:01:24.530 between gram-negative and gram-positive. 00:01:24.530 --> 00:01:27.470 So we were talking, at the end of the class, 00:01:27.470 --> 00:01:29.930 about, this was for the siderophores 00:01:29.930 --> 00:01:30.980 which we talked about. 00:01:30.980 --> 00:01:33.290 We need to take them up. 00:01:33.290 --> 00:01:36.470 These are common to all uptake systems. 00:01:36.470 --> 00:01:41.255 You have some kind of ATPase system and ABC ATPase. 00:01:41.255 --> 00:01:43.130 We're not going to talk about that in detail, 00:01:43.130 --> 00:01:47.540 but it uses ATP to bring these molecules 00:01:47.540 --> 00:01:52.620 and also heme molecules across the plasma membrane. 00:01:52.620 --> 00:01:55.340 And then, in all cases, you have this issue 00:01:55.340 --> 00:01:58.940 of how do you get the iron out of whatever the carrier is, 00:01:58.940 --> 00:02:03.320 be it a siderophore where the carriers can bind very tightly 00:02:03.320 --> 00:02:07.130 or heme where you also have to do something 00:02:07.130 --> 00:02:12.060 to get the iron out of the heme so that it can be used. 00:02:12.060 --> 00:02:17.280 And so what I want to just say, very briefly-- 00:02:17.280 --> 00:02:18.680 and this you all should know now. 00:02:18.680 --> 00:02:20.570 So now we're looking at heme uptake. 00:02:20.570 --> 00:02:24.110 I'm not going to spend a lot of time drawing the pictures out, 00:02:24.110 --> 00:02:30.530 but, if you look at the PowerPoint cartoon, what 00:02:30.530 --> 00:02:33.890 you will see is there is a protein like this, which 00:02:33.890 --> 00:02:36.840 hopefully you now have been introduced to from your problem 00:02:36.840 --> 00:02:37.340 set. 00:02:37.340 --> 00:02:43.130 So this could be IsdB or IsdH. 00:02:43.130 --> 00:02:46.640 And we'll come back to that, subsequently. 00:02:46.640 --> 00:02:51.080 And it sits on the outside of the peptidoglycan. 00:02:51.080 --> 00:02:53.150 So this is the protein. 00:02:53.150 --> 00:02:59.360 The key thing that is present in all these Isd proteins 00:02:59.360 --> 00:03:02.840 is-- let me draw this differently-- is a NEAT domain. 00:03:05.520 --> 00:03:06.020 OK? 00:03:06.020 --> 00:03:07.320 And we'll come back to that later on. 00:03:07.320 --> 00:03:08.060 But this domain-- 00:03:08.060 --> 00:03:09.643 So you have a big protein, and there's 00:03:09.643 --> 00:03:12.860 one little domain that's going to suck the heme out. 00:03:12.860 --> 00:03:16.910 And so what happens is we'll see in Staph. aureus, which 00:03:16.910 --> 00:03:20.650 is what we're going to be focused on, 00:03:20.650 --> 00:03:24.170 you have hemoglobin. 00:03:24.170 --> 00:03:28.160 And somehow-- and I'm going to indicate heme 00:03:28.160 --> 00:03:31.640 as a ball of orange, with a little planar 00:03:31.640 --> 00:03:33.470 thing as the protoporphyrin IX. 00:03:33.470 --> 00:03:35.510 OK, are you all with me? 00:03:35.510 --> 00:03:38.900 And then somehow this gets sucked out 00:03:38.900 --> 00:03:41.620 into the NEAT domain, where-- 00:03:41.620 --> 00:03:44.600 And again, all of these gram-positive and gram-negative 00:03:44.600 --> 00:03:48.890 systems are slightly different, but in the Staph. aureus system 00:03:48.890 --> 00:03:50.390 we'll be talking about today and you 00:03:50.390 --> 00:03:52.940 had to think about in the problem set you basically 00:03:52.940 --> 00:03:56.960 have a cascade of proteins which have additional NEAT 00:03:56.960 --> 00:04:03.470 domains from which, because this is such a large peptidoglycan, 00:04:03.470 --> 00:04:08.270 you need to transfer the heme to the plasma-membrane 00:04:08.270 --> 00:04:09.120 transporter. 00:04:09.120 --> 00:04:13.160 And what's interesting about these systems 00:04:13.160 --> 00:04:16.459 and is distinct is that they end up, 00:04:16.459 --> 00:04:23.950 they're covalently bound to the peptidoglycan. 00:04:23.950 --> 00:04:26.360 And I'm going to indicate peptidoglycan as "PG." 00:04:26.360 --> 00:04:29.050 And we'll talk about that reaction today-- 00:04:29.050 --> 00:04:32.200 the enzyme that catalyzes those reactions. 00:04:32.200 --> 00:04:35.740 And all of these guys end up covalently bound 00:04:35.740 --> 00:04:38.410 to the peptidoglycan-- which is distinct from all 00:04:38.410 --> 00:04:41.530 of the experiments you looked at in your problem set. 00:04:41.530 --> 00:04:44.590 Nobody can figure out how to make the peptidoglycan 00:04:44.590 --> 00:04:46.240 with these things covalently bound. 00:04:46.240 --> 00:04:51.070 So what you're looking at is a model for the actual process. 00:04:51.070 --> 00:04:55.630 OK, so, also-- so that's the gram-positive. 00:04:55.630 --> 00:05:01.100 And in the gram-negative, one has two ways of doing this. 00:05:01.100 --> 00:05:05.350 And again, these parallel the ways with siderophore uptake. 00:05:05.350 --> 00:05:09.770 So you have an outer membrane-- 00:05:09.770 --> 00:05:11.890 So this is the outer membrane. 00:05:11.890 --> 00:05:18.190 And you have a beta barrel, with a little plug in it. 00:05:18.190 --> 00:05:21.070 And so these beta barrels, they're at, like, 20 or 30 00:05:21.070 --> 00:05:23.410 of these things in the outer membranes. 00:05:23.410 --> 00:05:25.180 And they can take up siderophores, 00:05:25.180 --> 00:05:27.700 as we talked about last time, but they can also 00:05:27.700 --> 00:05:28.600 take up hemes. 00:05:28.600 --> 00:05:29.100 OK? 00:05:29.100 --> 00:05:30.750 So each one of these is distinct, 00:05:30.750 --> 00:05:34.930 although the structures are all pretty much the same. 00:05:34.930 --> 00:05:38.710 And so what you see in this case is, 00:05:38.710 --> 00:05:46.360 there are actually two ways that you can take heme up. 00:05:46.360 --> 00:05:50.050 So you can take up heme directly. 00:05:50.050 --> 00:05:53.320 And we'll see that what we'll be looking at 00:05:53.320 --> 00:05:58.180 is hemoglobin, which has four alpha 2 beta 2. 00:05:58.180 --> 00:06:00.550 So this could be hemoglobin. 00:06:00.550 --> 00:06:03.310 That's one of the major sources, and it is the major source 00:06:03.310 --> 00:06:05.470 for Staph. aureus. 00:06:05.470 --> 00:06:10.270 And so this can bind directly to the beta barrel-- 00:06:10.270 --> 00:06:12.060 gets extracted. 00:06:12.060 --> 00:06:13.420 The heme gets extracted. 00:06:13.420 --> 00:06:15.250 The protein doesn't get through. 00:06:15.250 --> 00:06:21.030 And so the heme is transferred through this beta barrel. 00:06:21.030 --> 00:06:22.210 OK. 00:06:22.210 --> 00:06:24.130 So that's one mechanism. 00:06:24.130 --> 00:06:27.070 And then there's a second mechanism. 00:06:27.070 --> 00:06:30.970 And the second mechanism involves a hemophore. 00:06:34.800 --> 00:06:40.660 And the hemophore is going to pick up the heme. 00:06:40.660 --> 00:06:43.960 And so every organism is distinct. 00:06:43.960 --> 00:06:46.690 There are many kinds of hemophores. 00:06:46.690 --> 00:06:50.260 And I have a definition of all of these-- 00:06:50.260 --> 00:06:52.240 the nomenclature involved. 00:06:52.240 --> 00:06:55.600 And so, after class today, I'll update these notes, 00:06:55.600 --> 00:06:57.340 because that's not in the original-- 00:06:57.340 --> 00:07:01.060 the definitions aren't in the original PowerPoint. 00:07:01.060 --> 00:07:01.780 OK? 00:07:01.780 --> 00:07:06.220 So what you have, over here, is the hemophore 00:07:06.220 --> 00:07:13.240 that somehow extracts the heme out 00:07:13.240 --> 00:07:15.370 of hemoglobin or haptoglobin. 00:07:15.370 --> 00:07:16.840 We'll see that's another thing. 00:07:16.840 --> 00:07:20.650 So this gets extracted and then gets 00:07:20.650 --> 00:07:23.290 transferred, in that fashion. 00:07:23.290 --> 00:07:27.280 And so these hemophores come in all flavors and shapes. 00:07:27.280 --> 00:07:30.590 They're different-- for example, in Pseudomonas or M. 00:07:30.590 --> 00:07:31.570 tuberculosis. 00:07:31.570 --> 00:07:34.000 And we're not going to talk about them further, 00:07:34.000 --> 00:07:39.220 but the idea is they all use these beta-barrel proteins 00:07:39.220 --> 00:07:43.630 to be able to somehow transfer the heme across. 00:07:43.630 --> 00:07:46.240 And what happens, just as in the case-- if you go back 00:07:46.240 --> 00:07:48.340 and you look at your notes from last time, 00:07:48.340 --> 00:07:50.740 there's a periplasmic binding protein 00:07:50.740 --> 00:07:54.790 that takes the heme and shuttles it, again, 00:07:54.790 --> 00:07:57.230 to these ABC transporters. 00:07:57.230 --> 00:07:57.730 OK? 00:07:57.730 --> 00:08:00.610 So, in this system, again, you have 00:08:00.610 --> 00:08:06.260 a periplasmic binding protein. 00:08:09.750 --> 00:08:19.000 And this goes to the ABC transporter, 00:08:19.000 --> 00:08:25.570 which uses ATP and the energy of hydrolysis of ATP, 00:08:25.570 --> 00:08:27.290 to transfer this into the cytosol. 00:08:30.090 --> 00:08:31.750 OK, so this is the same. 00:08:31.750 --> 00:08:34.210 That remains the same. 00:08:34.210 --> 00:08:37.539 And the transporters are distinct. 00:08:37.539 --> 00:08:40.240 And then, again, once you get inside the cell, 00:08:40.240 --> 00:08:41.590 what do you have to do? 00:08:41.590 --> 00:08:43.539 You've got to get the iron out of the heme. 00:08:43.539 --> 00:08:46.100 So the problems that you're facing 00:08:46.100 --> 00:08:49.540 are very similar to the siderophores. 00:08:49.540 --> 00:08:53.030 So, in all cases-- 00:08:53.030 --> 00:08:58.340 So the last step is, in the cytosol, 00:08:58.340 --> 00:09:05.590 you need to extract the iron. 00:09:05.590 --> 00:09:07.030 And you can extract-- 00:09:07.030 --> 00:09:10.630 usually, this is in a plus-3 oxidation state. 00:09:10.630 --> 00:09:12.910 So you extract the iron. 00:09:12.910 --> 00:09:21.350 And this can be done by a heme oxygenase, which 00:09:21.350 --> 00:09:23.320 degrades the heme. 00:09:23.320 --> 00:09:23.820 OK. 00:09:26.960 --> 00:09:29.240 In some cases, people have reported 00:09:29.240 --> 00:09:32.510 that you can reduce the iron 3 to iron 2, when the heme can 00:09:32.510 --> 00:09:35.570 come out, but that still probably is not an easy task 00:09:35.570 --> 00:09:37.400 because you've got four-- 00:09:37.400 --> 00:09:40.820 you've got four nitrogens, chelating to the heme, 00:09:40.820 --> 00:09:42.890 and the exchange, the ligand exchange, rates 00:09:42.890 --> 00:09:44.510 are probably really slow. 00:09:44.510 --> 00:09:47.690 So I would say the major way of getting 00:09:47.690 --> 00:09:50.630 the iron out of the heme is by degradation of the heme. 00:09:50.630 --> 00:09:54.710 And we're not going to talk about that in detail at all, 00:09:54.710 --> 00:09:55.250 either. 00:09:55.250 --> 00:09:56.450 OK. 00:09:56.450 --> 00:09:58.820 So that's the introductory part. 00:09:58.820 --> 00:10:00.380 And here's the nomenclature, which 00:10:00.380 --> 00:10:01.860 I've already gone through. 00:10:01.860 --> 00:10:03.920 I've got all these terms defined. 00:10:03.920 --> 00:10:06.710 And if you don't remember that, or you don't remember it 00:10:06.710 --> 00:10:09.920 from the reading, you have a page with all the names-- 00:10:09.920 --> 00:10:12.050 which are confusing. 00:10:12.050 --> 00:10:14.390 And so the final thing I wanted to say, 00:10:14.390 --> 00:10:19.550 before we go on and actually start looking at peptidoglycans 00:10:19.550 --> 00:10:21.740 and gram-positive bacteria and heme uptake 00:10:21.740 --> 00:10:25.310 in Staph. aureus, which is what I was going to focus on 00:10:25.310 --> 00:10:29.090 in this little module, is to just show you, 00:10:29.090 --> 00:10:32.010 bacteria desperately need iron. 00:10:32.010 --> 00:10:33.050 So what do they do? 00:10:33.050 --> 00:10:34.700 This is what they do. 00:10:34.700 --> 00:10:39.320 OK, so, here you can see-- and some bacteria 00:10:39.320 --> 00:10:41.900 make three or four kinds of siderophores. 00:10:41.900 --> 00:10:44.870 Others only make one or two kinds of siderophores, 00:10:44.870 --> 00:10:46.790 but what they've done is they've figured out 00:10:46.790 --> 00:10:51.560 how to scavenge the genes that are required 00:10:51.560 --> 00:10:52.970 for these beta barrels. 00:10:52.970 --> 00:10:54.830 So they can take up a siderophore 00:10:54.830 --> 00:10:57.120 that some other bacteria makes. 00:10:57.120 --> 00:10:57.620 OK? 00:10:57.620 --> 00:10:59.330 And that's also true of yeast. 00:10:59.330 --> 00:11:03.410 Yeast don't make siderophores, but most yeast have, 00:11:03.410 --> 00:11:05.870 in their outer membranes, ways of picking up 00:11:05.870 --> 00:11:08.708 siderophores and bringing it into the cell, since-- 00:11:08.708 --> 00:11:10.250 and remember we talked about the fact 00:11:10.250 --> 00:11:13.610 there were 500 different kinds of siderophores. 00:11:13.610 --> 00:11:16.890 But you can see that the strategy is exactly the same. 00:11:16.890 --> 00:11:18.920 You have a beta barrel. 00:11:18.920 --> 00:11:22.670 You have-- these are all periplasmic binding proteins. 00:11:22.670 --> 00:11:26.840 This picture is screwed up, in that they forgot the TonB. 00:11:26.840 --> 00:11:31.250 Remember, there's a three-component machine, 00:11:31.250 --> 00:11:37.100 TonB, ExbB and D, which is connected to a proton motive 00:11:37.100 --> 00:11:39.680 force across a plasma membrane, which 00:11:39.680 --> 00:11:44.630 is key for getting either the heme or the iron 00:11:44.630 --> 00:11:46.860 into the periplasm. 00:11:46.860 --> 00:11:49.830 And you use a periplasmic binding protein, 00:11:49.830 --> 00:11:54.410 which then goes through these ATPase transp-- 00:11:54.410 --> 00:11:57.230 ABC-ATPase transporters. 00:11:57.230 --> 00:12:02.090 So what I showed you was heme uptake, iron uptake, but in all 00:12:02.090 --> 00:12:06.380 of these cases, like Staph. aureus we'll be talking about, 00:12:06.380 --> 00:12:08.590 we can also get iron out of transferrin. 00:12:08.590 --> 00:12:09.590 We've talked about that. 00:12:09.590 --> 00:12:11.660 That's the major carrier in humans. 00:12:11.660 --> 00:12:15.830 The siderophores can actually extract the iron 00:12:15.830 --> 00:12:16.940 from the transferrin. 00:12:16.940 --> 00:12:19.130 And remember the KD was 10 to the minus 3, 00:12:19.130 --> 00:12:22.400 so somehow, again, you've got to get iron transferred 00:12:22.400 --> 00:12:24.410 under those conditions. 00:12:24.410 --> 00:12:26.090 And that's how these guys survive. 00:12:26.090 --> 00:12:32.140 So they're pretty desperate to get iron. 00:12:32.140 --> 00:12:34.340 And inside, once they get inside the cell, 00:12:34.340 --> 00:12:38.137 you have all variations of the theme to get the iron out. 00:12:38.137 --> 00:12:39.470 But they're all sort of similar. 00:12:39.470 --> 00:12:42.012 Somehow, you've got to get rid of whatever is tightly binding 00:12:42.012 --> 00:12:43.070 it. 00:12:43.070 --> 00:12:46.370 And if you're creative, you can reuse 00:12:46.370 --> 00:12:50.010 whatever is tightly binding it, to go pick up some more metal. 00:12:50.010 --> 00:12:50.510 OK. 00:12:50.510 --> 00:12:53.630 So that just summarizes what I just said. 00:12:53.630 --> 00:12:56.060 And so, in two seconds, I'm going to show you, 00:12:56.060 --> 00:12:57.740 now-- we've spent one whole lecture, 00:12:57.740 --> 00:12:59.282 a little more than a lecture, talking 00:12:59.282 --> 00:13:03.650 about iron uptake in humans via DMT1, 00:13:03.650 --> 00:13:08.900 the iron-2 transporter, and the transferrin transfer receptor. 00:13:08.900 --> 00:13:11.390 So, in the plus-two and plus-three states, 00:13:11.390 --> 00:13:15.500 we just started looking at the strategies by bacteria 00:13:15.500 --> 00:13:18.560 and saw how widespread they are. 00:13:18.560 --> 00:13:22.700 And then the question is, how do you win? 00:13:22.700 --> 00:13:26.000 OK, bacteria need iron. 00:13:26.000 --> 00:13:27.530 We need iron. 00:13:27.530 --> 00:13:29.750 And the question is, how do you reach-- 00:13:29.750 --> 00:13:31.950 and we have a lot of bacteria growing in us, [LAUGH] 00:13:31.950 --> 00:13:35.210 so we've reached some kind of homeostasis. 00:13:35.210 --> 00:13:37.010 But with the pathogenic ones, of course, 00:13:37.010 --> 00:13:39.050 we really want to get rid of them. 00:13:39.050 --> 00:13:40.640 And so that's what the issue is. 00:13:40.640 --> 00:13:44.030 And there have been a bunch of articles. 00:13:44.030 --> 00:13:46.010 You can read about this in a lot of detail, 00:13:46.010 --> 00:13:50.270 if you're interested in the more medical aspects of this. 00:13:50.270 --> 00:13:53.570 But this war between bacteria and humans. 00:13:53.570 --> 00:13:56.230 And really it's sort of fight for nutrients. 00:13:56.230 --> 00:13:59.450 And, in this case, the nutrient is iron. 00:13:59.450 --> 00:14:03.050 Has received a lot of attention, because we're 00:14:03.050 --> 00:14:05.870 desperate for new kinds of targets 00:14:05.870 --> 00:14:08.720 for antibiotics, because of the resistance problems. 00:14:08.720 --> 00:14:14.150 And so nutrient limitation and iron sequestration 00:14:14.150 --> 00:14:18.288 from a pathogenic organism might represent a new target. 00:14:18.288 --> 00:14:19.580 Of course, what are the issues? 00:14:19.580 --> 00:14:22.040 The issues are, we also need iron. 00:14:22.040 --> 00:14:24.457 And so, if you lower the amount of iron, 00:14:24.457 --> 00:14:26.040 then you might be in trouble, as well. 00:14:26.040 --> 00:14:29.860 So what we know is, bacteria, viruses-- 00:14:29.860 --> 00:14:32.130 bacteria have been extensively studied; 00:14:32.130 --> 00:14:40.080 viruses, less so, also protozoa, such as the malaria system-- 00:14:40.080 --> 00:14:44.160 all are known to depend on iron for growth. 00:14:44.160 --> 00:14:47.370 And so, again, if you want to read about this, 00:14:47.370 --> 00:14:49.620 you can read about some of the strategies 00:14:49.620 --> 00:14:52.290 these organisms [LAUGH] use to get iron away 00:14:52.290 --> 00:14:54.443 from the human systems. 00:14:54.443 --> 00:14:55.860 And it's sort of amazing, when you 00:14:55.860 --> 00:14:58.320 look at the details of how things have evolved, 00:14:58.320 --> 00:15:03.010 back and forth, back and forth, [LAUGH] in terms of survival. 00:15:03.010 --> 00:15:05.980 And so really what it's all about is homeostasis. 00:15:05.980 --> 00:15:06.810 OK? 00:15:06.810 --> 00:15:09.100 And that's what was all about in cholesterol. 00:15:09.100 --> 00:15:11.430 And we'll see, with reactive oxygen species, 00:15:11.430 --> 00:15:13.650 that's what it's all about. 00:15:13.650 --> 00:15:20.020 So, somehow, using hepcidin-- 00:15:20.020 --> 00:15:21.900 which is the human master regulator, 00:15:21.900 --> 00:15:23.670 the peptide hormone-- 00:15:23.670 --> 00:15:26.190 we need to figure out how to keep ourselves alive 00:15:26.190 --> 00:15:30.180 while killing off these bacteria, in some way, 00:15:30.180 --> 00:15:34.070 by sequestering the iron from the bacteria. 00:15:34.070 --> 00:15:34.570 OK. 00:15:34.570 --> 00:15:37.050 So this is an important problem that has 00:15:37.050 --> 00:15:38.370 received a lot of attention. 00:15:38.370 --> 00:15:42.570 And most of you know that the Nolan Lab is doing 00:15:42.570 --> 00:15:44.940 beautiful studies in this area. 00:15:44.940 --> 00:15:45.690 OK. 00:15:45.690 --> 00:15:48.390 So what I want to do now, for the rest of the lecture, 00:15:48.390 --> 00:15:50.430 is focus on Staph. aureus. 00:15:50.430 --> 00:15:51.960 OK? 00:15:51.960 --> 00:15:57.270 And Staph. aureus is-- 00:15:57.270 --> 00:16:02.550 methicillin-resistant Staph. aureus is a major problem, 00:16:02.550 --> 00:16:04.050 throughout the world. 00:16:04.050 --> 00:16:06.240 We don't have any ways to kill this guy. 00:16:06.240 --> 00:16:08.880 And so that's why I decided to pick this target, 00:16:08.880 --> 00:16:12.240 but there are many other [LAUGH] of these pathogens around that 00:16:12.240 --> 00:16:14.460 have problems-- 00:16:14.460 --> 00:16:18.630 have also resistance problems, Staph. aureus 00:16:18.630 --> 00:16:21.120 being the one that's been most extensively studied 00:16:21.120 --> 00:16:24.000 in the last decade or so. 00:16:24.000 --> 00:16:28.100 But bacteria has come back in vogue. 00:16:28.100 --> 00:16:31.570 For years, nobody on campus cared anything about [LAUGH] 00:16:31.570 --> 00:16:33.540 microorganisms or bacteria. 00:16:33.540 --> 00:16:36.283 The microbiome has brought it back in vogue, 00:16:36.283 --> 00:16:37.950 because people think they're going to be 00:16:37.950 --> 00:16:39.240 able to figure that all out. 00:16:39.240 --> 00:16:40.260 OK. 00:16:40.260 --> 00:16:43.880 But anyhow, bacteria have always been extremely important, 00:16:43.880 --> 00:16:45.540 not only in terms of human health 00:16:45.540 --> 00:16:49.002 but in terms of how the whole world functions. 00:16:49.002 --> 00:16:50.460 There are so many of them, and they 00:16:50.460 --> 00:16:52.680 do so much interesting stuff. 00:16:52.680 --> 00:16:54.690 And we have to live with them, side by side. 00:16:54.690 --> 00:16:58.568 So anyhow, we're going to look at Staph. aureus. 00:16:58.568 --> 00:17:01.110 That's what we're going to focus on, because of this problem. 00:17:01.110 --> 00:17:04.650 And I think Staph. aureus, which many people don't realize, 00:17:04.650 --> 00:17:08.940 is that 30% of all people have Staph. aureus on your skin 00:17:08.940 --> 00:17:13.690 or in regions that are not breaching into the bloodstream. 00:17:13.690 --> 00:17:15.560 So we all have Staph. aureus. 00:17:15.560 --> 00:17:21.300 So 30% of us have this bacteria. 00:17:21.300 --> 00:17:25.560 If you get-- if wherever it's localized is breached, 00:17:25.560 --> 00:17:28.349 and it gets into our bloodstream, 00:17:28.349 --> 00:17:30.240 then it's all over, because Staph. aureus 00:17:30.240 --> 00:17:31.890 can colonize almost anywhere. 00:17:31.890 --> 00:17:33.870 That's different from other organisms. 00:17:33.870 --> 00:17:36.300 Some organisms can only colonize in the lungs. 00:17:36.300 --> 00:17:38.200 Some colonize in the heart. 00:17:38.200 --> 00:17:44.590 So these can colonize almost all tissues. 00:17:44.590 --> 00:17:46.320 And what you know is, if you start 00:17:46.320 --> 00:17:49.320 thinking about physiology-- and again, I'm not an MD-- 00:17:49.320 --> 00:17:52.270 but different tissues have different environments. 00:17:52.270 --> 00:17:52.900 OK? 00:17:52.900 --> 00:17:55.650 And so a lot of organisms find siderophore an environment 00:17:55.650 --> 00:18:01.260 where they can best live and then take up-- 00:18:01.260 --> 00:18:02.730 make their home there. 00:18:02.730 --> 00:18:04.590 But Staph. aureus is one of these guys 00:18:04.590 --> 00:18:07.050 that can go anywhere. 00:18:07.050 --> 00:18:12.410 And so this makes it specifically very insidious. 00:18:12.410 --> 00:18:18.560 And you can get septicemia, or you can get endocarditis, 00:18:18.560 --> 00:18:22.520 or you can get all kinds of horrible diseases associated 00:18:22.520 --> 00:18:26.310 with Staph. aureus, once it breaches the barrier. 00:18:26.310 --> 00:18:26.810 OK. 00:18:26.810 --> 00:18:31.850 So what we need to do, as you've already seen from your problem 00:18:31.850 --> 00:18:38.020 set, to understand how Staph. aureus can get heme 00:18:38.020 --> 00:18:45.570 into its cytosol to be able to function, 00:18:45.570 --> 00:18:48.320 to be able to grow effectively, is, 00:18:48.320 --> 00:18:51.380 we need to look at the outer cell wall or the peptidoglycan. 00:18:51.380 --> 00:18:55.370 So what I'm going to do is spend a few minutes 00:18:55.370 --> 00:18:58.520 talking about the structure of the peptidoglycan. 00:18:58.520 --> 00:19:00.140 And then we'll go back in and we'll 00:19:00.140 --> 00:19:04.250 talk about how these proteins that you worked on 00:19:04.250 --> 00:19:07.390 in the problem set covalently bind to the peptidoglycan 00:19:07.390 --> 00:19:09.920 and allow you to take up iron to the cell. 00:19:09.920 --> 00:19:13.250 And why is heme a major target? 00:19:13.250 --> 00:19:15.740 Heme is a major target for Staph. aureus. 00:19:15.740 --> 00:19:17.420 They've evolved. 00:19:17.420 --> 00:19:19.810 The major source of iron, we all know, 00:19:19.810 --> 00:19:22.580 is hemoglobin now, in red blood cells. 00:19:22.580 --> 00:19:26.270 And so Staph. aureus has developed proteins-- 00:19:26.270 --> 00:19:29.150 endotoxins, really-- that can go in 00:19:29.150 --> 00:19:34.070 and-- there's proteins that can insert into red blood cell 00:19:34.070 --> 00:19:36.580 membranes, make a pore. 00:19:36.580 --> 00:19:40.730 The blood cells lyse, and now the bacteria 00:19:40.730 --> 00:19:43.640 are extremely happy because they have huge amounts of heme. 00:19:43.640 --> 00:19:47.930 And then they want to take that heme into-- 00:19:47.930 --> 00:19:49.320 to help them survive. 00:19:49.320 --> 00:19:52.370 So Staph. aureus are amazingly creative, 00:19:52.370 --> 00:19:57.610 in terms of getting the heme that they need for survival. 00:19:57.610 --> 00:19:58.110 OK. 00:19:58.110 --> 00:20:00.380 So, peptidoglycan. 00:20:00.380 --> 00:20:03.440 Most of you have probably seen peptidoglycan before. 00:20:03.440 --> 00:20:07.590 I'm just going to say a few things about peptidoglycan. 00:20:07.590 --> 00:20:09.700 So let's look at-- 00:20:09.700 --> 00:20:10.200 let's see. 00:20:10.200 --> 00:20:11.190 Where do I want to do this? 00:20:11.190 --> 00:20:11.690 All right. 00:20:11.690 --> 00:20:13.060 So I'm going to erase this. 00:20:13.060 --> 00:20:15.900 We're going to look at the cell wall. 00:20:15.900 --> 00:20:16.400 OK. 00:20:20.120 --> 00:20:23.150 And what you can see, here, I'm going to draw just a few things 00:20:23.150 --> 00:20:24.030 on the board. 00:20:24.030 --> 00:20:26.810 But what you can see here, in this cartoon, 00:20:26.810 --> 00:20:29.720 is you have two kinds of sugars-- 00:20:29.720 --> 00:20:33.500 N-acetylglucosamine and N-acetylmuramic acid. 00:20:33.500 --> 00:20:36.020 N-acetylglucosamine is a precursor 00:20:36.020 --> 00:20:38.780 to N-acetylmuramic acid. 00:20:38.780 --> 00:20:42.530 And what you see, attached to N-acetylmuramic acid, 00:20:42.530 --> 00:20:44.270 are little blue balls. 00:20:44.270 --> 00:20:46.280 And that's the peptide that turns out 00:20:46.280 --> 00:20:47.840 it starts out with a pentapeptide 00:20:47.840 --> 00:20:50.540 and goes to a tetrapeptide. 00:20:50.540 --> 00:20:53.520 And what you see here, in the purple balls-- 00:20:53.520 --> 00:20:57.170 and this is unique to Staph. aureus-- 00:20:57.170 --> 00:21:00.330 is, other amino acids, they're all the same, 00:21:00.330 --> 00:21:01.250 and this is glycine. 00:21:01.250 --> 00:21:06.080 So, if you look down here, here are the disaccharides, shown up 00:21:06.080 --> 00:21:07.430 here. 00:21:07.430 --> 00:21:10.670 Here is-- yeah, one, two, three, four, five. 00:21:10.670 --> 00:21:12.870 Here is the pentapeptide. 00:21:12.870 --> 00:21:16.800 And what do you notice unusual about the pentapeptide? 00:21:16.800 --> 00:21:19.340 You have a D glutamine. 00:21:19.340 --> 00:21:20.060 OK? 00:21:20.060 --> 00:21:22.580 And I was just reading a whole bunch of papers 00:21:22.580 --> 00:21:23.660 on somebody's thesis-- 00:21:23.660 --> 00:21:26.012 tomorrow, actually. 00:21:26.012 --> 00:21:27.470 And you're trying to make this guy, 00:21:27.470 --> 00:21:28.920 nobody can study this stuff. 00:21:28.920 --> 00:21:29.630 Why? 00:21:29.630 --> 00:21:32.550 Because you have to make a peptidoglycan. 00:21:32.550 --> 00:21:33.300 And I'll show you. 00:21:33.300 --> 00:21:34.130 It's complicated. 00:21:34.130 --> 00:21:36.360 You have to stick on a pentapeptide. 00:21:36.360 --> 00:21:38.905 You have to stick on the glycines. 00:21:38.905 --> 00:21:40.280 And how do you get the substrates 00:21:40.280 --> 00:21:42.110 for your enzymatic reactions? 00:21:42.110 --> 00:21:44.570 So we've known this pathway for decades, 00:21:44.570 --> 00:21:46.190 but it's taken really good chemists 00:21:46.190 --> 00:21:50.660 to be able to figure out how to look at these individual steps. 00:21:50.660 --> 00:21:54.740 And so what's unusual, here, is, if you replace glutamine 00:21:54.740 --> 00:21:58.490 with a glutamate, it doesn't work very well at all. 00:21:58.490 --> 00:22:00.530 OK, so it's that subtle. 00:22:00.530 --> 00:22:02.840 Here you've got this huge macromolecule, 00:22:02.840 --> 00:22:05.660 and you're replacing an NH2 with an OH, 00:22:05.660 --> 00:22:09.920 and you alter the resistance to different bacteria. 00:22:09.920 --> 00:22:13.130 And again, you have this unusual pentaglycine. 00:22:13.130 --> 00:22:16.610 And you'll see in the cartoon, in a few minutes, 00:22:16.610 --> 00:22:20.280 where do you think this glycine, pentaglycine comes from? 00:22:20.280 --> 00:22:23.910 Well, it actually comes from a tRNA that binds glycine. 00:22:23.910 --> 00:22:25.980 OK, you've seen that before. 00:22:25.980 --> 00:22:28.430 But, instead of using the ribosome 00:22:28.430 --> 00:22:33.230 to make this little peptide, it uses nonribosomal peptide 00:22:33.230 --> 00:22:34.520 synthetases. 00:22:34.520 --> 00:22:37.520 And this all happens in the cytosol of the cell. 00:22:37.520 --> 00:22:41.300 So, what do we know about the structure? 00:22:41.300 --> 00:22:47.330 I'm just going to draw N-acetylglucosamine. 00:22:47.330 --> 00:22:51.680 And what I'm going to do is put some R groups on here. 00:22:51.680 --> 00:22:53.960 So I'm going to put OX. 00:22:53.960 --> 00:22:57.680 And then here we have N-acetyl. 00:22:57.680 --> 00:23:01.040 So that's an acetate group. 00:23:01.040 --> 00:23:03.545 Here I'm going to put another OR group. 00:23:08.740 --> 00:23:13.600 OK, so the two things I want to focus on, 00:23:13.600 --> 00:23:17.800 the two things I'm going to focus on, is this X 00:23:17.800 --> 00:23:21.677 and this R. So is N-acetylglucosamine. 00:23:24.790 --> 00:23:31.570 And then the second one is N-acetylmuramic acid. 00:23:31.570 --> 00:23:39.172 And, in both of these cases, X is equal to UDP. 00:23:39.172 --> 00:23:40.630 So we're going to come back to this 00:23:40.630 --> 00:23:43.120 in the last module on nucleotides. 00:23:43.120 --> 00:23:47.620 So nucleotides play a central role in RNA and DNA, 00:23:47.620 --> 00:23:49.750 but they also play a central role 00:23:49.750 --> 00:23:53.290 in moving around all sugars inside the cell. 00:23:53.290 --> 00:23:57.640 So what you have here, actually, is a pyrophosphate linkage 00:23:57.640 --> 00:23:59.130 to UDP. 00:23:59.130 --> 00:24:00.430 OK? 00:24:00.430 --> 00:24:04.370 And if we look at N-acetylglucosamine, 00:24:04.370 --> 00:24:07.360 R is equal to H. OK? 00:24:07.360 --> 00:24:10.720 But if we look at muramic acid, what we're going to see 00:24:10.720 --> 00:24:15.565 is that nature has put on a lactic acid in this position. 00:24:18.780 --> 00:24:23.040 OK, so here's your methyl group, from your lactic acid. 00:24:23.040 --> 00:24:24.860 And here's the carboxylate. 00:24:24.860 --> 00:24:32.570 So this is the R group in N-acetylmuramic acid. 00:24:32.570 --> 00:24:33.440 OK. 00:24:33.440 --> 00:24:35.930 Now, what we're going to see is, while most sugars-- 00:24:35.930 --> 00:24:41.390 and this is true in humans, and it's also true in bacteria-- 00:24:41.390 --> 00:24:44.540 are carried around and transported within the cell 00:24:44.540 --> 00:24:48.770 as linked nucleotides, what we'll also see in the cell 00:24:48.770 --> 00:24:53.690 wall-- which has made them extremely challenging to study, 00:24:53.690 --> 00:24:57.380 made the whole pathway extremely challenging to study-- 00:24:57.380 --> 00:25:03.200 in addition to X equal to UDP, X can also 00:25:03.200 --> 00:25:11.240 be equal to sort of an amazing structure. 00:25:11.240 --> 00:25:13.120 And the structure is slightly different 00:25:13.120 --> 00:25:16.490 in different bacteria, but this strategy 00:25:16.490 --> 00:25:22.820 is also used in humans, where you have a lipid 00:25:22.820 --> 00:25:26.225 and you have a lipid that acts as-- 00:25:29.390 --> 00:25:31.820 is made from-- hopefully you now know-- 00:25:31.820 --> 00:25:34.120 is isopentenyl pyrophosphate. 00:25:34.120 --> 00:25:35.610 OK? 00:25:35.610 --> 00:25:37.730 And there are seven of these, where you 00:25:37.730 --> 00:25:40.460 have the trans configuration. 00:25:40.460 --> 00:25:42.710 There are now three of these, which 00:25:42.710 --> 00:25:45.290 have the cis configuration. 00:25:48.260 --> 00:25:51.895 Just make sure I get my-- 00:25:51.895 --> 00:25:54.190 is that right? 00:25:54.190 --> 00:25:55.192 Yeah, that's right. 00:25:55.192 --> 00:25:56.650 OK, so you have three of these that 00:25:56.650 --> 00:25:58.520 have the cis configuration. 00:25:58.520 --> 00:26:02.770 And then you have a terminal dimethyl L configuration. 00:26:02.770 --> 00:26:05.590 And this is C55. 00:26:05.590 --> 00:26:07.840 So, if you're a synthetic chemist, 00:26:07.840 --> 00:26:11.860 and you're trying to stick on a couple of these sugars 00:26:11.860 --> 00:26:14.200 with hydrocarbon on the tail, with C55, 00:26:14.200 --> 00:26:17.590 you can imagine you would have one heck of a trouble, number 00:26:17.590 --> 00:26:20.890 one, synthesizing it but, number two, dealing with it. 00:26:20.890 --> 00:26:23.200 And so this goes to the question which 00:26:23.200 --> 00:26:24.850 I think is really interesting is, 00:26:24.850 --> 00:26:27.880 many people think about polymerization reactions. 00:26:27.880 --> 00:26:31.240 We're going to see this polymer is non-template-dependent, 00:26:31.240 --> 00:26:35.320 in contrast to polymers of DNA RA, where you have a template. 00:26:35.320 --> 00:26:38.990 And furthermore, DNA and RNA are pretty soluble. 00:26:38.990 --> 00:26:40.930 These things become insoluble. 00:26:40.930 --> 00:26:45.220 So you're making a phase transition from soluble state 00:26:45.220 --> 00:26:48.250 to an insoluble state, around the bacteria. 00:26:48.250 --> 00:26:52.060 And I think it's really sort of a tribute to Strominger, who 00:26:52.060 --> 00:26:54.370 worked on this many years ago, that he figured out 00:26:54.370 --> 00:26:55.460 sort of the pathway. 00:26:55.460 --> 00:26:59.050 But now it's only with recent studies, and really 00:26:59.050 --> 00:27:01.060 some very hard work synthetically, 00:27:01.060 --> 00:27:07.240 and also in terms of the microbiology and biochemistry, 00:27:07.240 --> 00:27:10.120 that it's really allowed us to elucidate this. 00:27:10.120 --> 00:27:16.480 So X, in this case, can also be this lipid. 00:27:16.480 --> 00:27:20.000 So I'm just pointing out what the issues are. 00:27:20.000 --> 00:27:23.450 And if you look at the cell wall, biosynthetic pathway-- 00:27:23.450 --> 00:27:25.840 so this is inside-- 00:27:25.840 --> 00:27:28.000 you're not going to be responsible for the details 00:27:28.000 --> 00:27:29.260 of this. 00:27:29.260 --> 00:27:31.930 But this is outside. 00:27:31.930 --> 00:27:32.430 OK. 00:27:32.430 --> 00:27:34.850 So you start out with a couple of sugars. 00:27:34.850 --> 00:27:37.960 These are the sugars we just talked about. 00:27:37.960 --> 00:27:38.480 OK. 00:27:38.480 --> 00:27:43.230 So now what you do is add on these five amino acids. 00:27:43.230 --> 00:27:45.440 So, over here, we ultimately need 00:27:45.440 --> 00:27:51.440 to add on five amino acids. 00:27:51.440 --> 00:27:54.380 And what do we see about the amino acids? 00:27:54.380 --> 00:27:57.470 They're unusual, because they can have the D-- they are not 00:27:57.470 --> 00:27:59.210 necessarily L-amino acids. 00:27:59.210 --> 00:28:01.490 They can be D-amino acids. 00:28:01.490 --> 00:28:03.170 And these things unfortunately are 00:28:03.170 --> 00:28:05.910 unique to different organisms. 00:28:05.910 --> 00:28:08.030 So, if you worked out a synthetic method for one, 00:28:08.030 --> 00:28:11.180 you're still faced with the problem that every one of them 00:28:11.180 --> 00:28:15.480 has different pentapeptides stuck on the end of it. 00:28:15.480 --> 00:28:17.180 Now, how would you attach-- 00:28:17.180 --> 00:28:20.420 you've now had a lot of biochemistry, 00:28:20.420 --> 00:28:21.950 where you've dealt with amino acids, 00:28:21.950 --> 00:28:23.720 in the first half of this course. 00:28:23.720 --> 00:28:27.770 How would you attach amino acids-- 00:28:27.770 --> 00:28:29.570 form and the linkages-- 00:28:29.570 --> 00:28:31.430 to this lactic acid? 00:28:31.430 --> 00:28:33.670 Can anybody tell me? 00:28:33.670 --> 00:28:36.312 What would you do, to make that attachment? 00:28:39.756 --> 00:28:43.690 AUDIENCE: You activate the carboxylate. 00:28:43.690 --> 00:28:46.190 JOANNE STUBBE: Yeah, so we have to activate the carboxylate. 00:28:46.190 --> 00:28:49.364 How do you activate the carboxylate? 00:28:49.364 --> 00:28:50.317 AUDIENCE: Make an AMP. 00:28:50.317 --> 00:28:51.150 JOANNE STUBBE: Yeah. 00:28:51.150 --> 00:28:54.350 So you make an AMP, just like you've seen with nonribosomal-- 00:28:54.350 --> 00:28:57.430 the adenlyating enzyme of nonribosomal polypeptide 00:28:57.430 --> 00:29:01.510 synthases, and you've seen with tRNAs. 00:29:01.510 --> 00:29:02.010 OK. 00:29:02.010 --> 00:29:05.920 So you see the same thing, over and over and over again. 00:29:05.920 --> 00:29:08.430 So you add these things on. 00:29:08.430 --> 00:29:11.310 The difference is that, again, these things, which 00:29:11.310 --> 00:29:13.770 are all soluble, down here, these are all 00:29:13.770 --> 00:29:15.870 soluble with the nucleotides. 00:29:15.870 --> 00:29:18.870 Now, because ultimately this needs 00:29:18.870 --> 00:29:20.370 to go from the inside of the cell 00:29:20.370 --> 00:29:23.670 to the outside of the cell, what you do, 00:29:23.670 --> 00:29:28.230 presumably, is take this lipid-- so you have the C55 lipid, 00:29:28.230 --> 00:29:30.690 with one phosphate on it. 00:29:30.690 --> 00:29:34.770 And then you attach it to one sugar. 00:29:34.770 --> 00:29:36.780 So here it's attached to the muramic acid, 00:29:36.780 --> 00:29:39.900 and that's called "lipid 1." 00:29:39.900 --> 00:29:43.110 You add N-acetylglucosamine with a glycosyltransferase. 00:29:43.110 --> 00:29:44.460 That's lipid 2. 00:29:44.460 --> 00:29:47.350 And that's the substrate for the polymerization reaction. 00:29:47.350 --> 00:29:48.390 What is the issue? 00:29:48.390 --> 00:29:50.760 The issue is, it's in the cytosol 00:29:50.760 --> 00:29:54.840 and all the chemistry happens on the outside of the cell. 00:29:54.840 --> 00:29:58.500 But, of course, if you move it from the inside to the outside, 00:29:58.500 --> 00:30:01.170 you don't want your substrates to float away. 00:30:01.170 --> 00:30:02.570 You've got to keep them there. 00:30:02.570 --> 00:30:03.120 OK? 00:30:03.120 --> 00:30:04.710 And that's especially [LAUGH] true 00:30:04.710 --> 00:30:09.040 in gram-positives, where we have no outer membrane. 00:30:09.040 --> 00:30:15.890 So the question is, how does this species get from this side 00:30:15.890 --> 00:30:18.280 to this side? 00:30:18.280 --> 00:30:18.780 OK. 00:30:18.780 --> 00:30:21.120 In the last couple years, people have proposed-- 00:30:21.120 --> 00:30:22.740 and so this has taken a long time. 00:30:22.740 --> 00:30:25.300 People have been looking for these proteins for decades. 00:30:25.300 --> 00:30:27.210 These are called "flipases." 00:30:27.210 --> 00:30:29.010 So you still have this issue-- again, 00:30:29.010 --> 00:30:32.808 this big, huge thing that needs to be transferred. 00:30:32.808 --> 00:30:34.350 And I think what's even more amazing, 00:30:34.350 --> 00:30:36.840 in the case of Staph. aureus, is that you 00:30:36.840 --> 00:30:39.270 put on the pentaglycine in the cytosol. 00:30:39.270 --> 00:30:43.050 So, here, what you'll see-- 00:30:43.050 --> 00:30:44.050 I think this is E. coli. 00:30:44.050 --> 00:30:45.760 I can't remember one from the other. 00:30:45.760 --> 00:30:51.190 But, instead of having DAP, which is diaminopimelate, 00:30:51.190 --> 00:30:52.680 you actually have lysine. 00:30:52.680 --> 00:30:58.020 So, here, what you have in Staph. aureus is a lysine, 00:30:58.020 --> 00:31:00.910 and the lysine has an amino group. 00:31:00.910 --> 00:31:06.182 And attached to this amino group is the pentaglycine. 00:31:06.182 --> 00:31:07.640 And this all occurs in the cytosol. 00:31:11.650 --> 00:31:13.790 So this is quite remarkable. 00:31:13.790 --> 00:31:20.260 So then, not only do you have to get the disaccharide 00:31:20.260 --> 00:31:22.390 with the pentapeptide on it, you need to have, 00:31:22.390 --> 00:31:24.650 here, the pentaglycine on it, as well. 00:31:24.650 --> 00:31:27.550 And this becomes really important in thinking 00:31:27.550 --> 00:31:32.910 about trying to study what's going on in the polymerization 00:31:32.910 --> 00:31:37.390 reaction, which is the target of natural products 00:31:37.390 --> 00:31:39.160 that are currently used, clinically. 00:31:39.160 --> 00:31:39.700 OK. 00:31:39.700 --> 00:31:41.680 So this thing's got to flip. 00:31:41.680 --> 00:31:43.840 And then what you have is a substrate. 00:31:43.840 --> 00:31:45.790 You have a growing chain. 00:31:45.790 --> 00:31:49.600 OK, and then what you need to do is extend this chain, 00:31:49.600 --> 00:31:51.440 so you have a glycosyltransferase. 00:31:51.440 --> 00:31:52.510 So you have two things. 00:31:52.510 --> 00:31:56.452 You have phosphoglycosyltransferase. 00:32:01.060 --> 00:32:04.540 And then the other thing you have is a TP, which 00:32:04.540 --> 00:32:05.860 is a transpeptidase. 00:32:10.280 --> 00:32:10.960 OK. 00:32:10.960 --> 00:32:13.150 And so the transpeptidase-- we're going to come back 00:32:13.150 --> 00:32:14.800 to this in a second, but-- 00:32:14.800 --> 00:32:19.390 is ultimately responsible for making a cross link. 00:32:19.390 --> 00:32:24.010 Which is what gives the bacteria cell wall rigidity. 00:32:24.010 --> 00:32:28.360 Now, in many organisms, the glycosyltransferase 00:32:28.360 --> 00:32:31.260 and the transpeptidase are on the same protein. 00:32:31.260 --> 00:32:32.800 They're two domains. 00:32:32.800 --> 00:32:37.320 But, in many organisms, they're not. 00:32:37.320 --> 00:32:42.340 OK, so you have two separate proteins. 00:32:42.340 --> 00:32:44.320 And furthermore, in Staph. aureus 00:32:44.320 --> 00:32:47.360 there are now five of these kinds of proteins. 00:32:47.360 --> 00:32:49.060 So the question is, what are all five 00:32:49.060 --> 00:32:51.460 of these glycosyltransferase doing? 00:32:51.460 --> 00:32:52.960 Which ones are involved in which? 00:32:52.960 --> 00:32:56.853 Which ones are involved in antibiotic resistance? 00:32:56.853 --> 00:32:59.020 And I think, when you start looking at it like this, 00:32:59.020 --> 00:33:00.550 you know, it's very complex. 00:33:00.550 --> 00:33:04.420 You realize what a hard problem this actually is. 00:33:04.420 --> 00:33:05.980 But we now have the tools, I think, 00:33:05.980 --> 00:33:07.360 because of beautiful studies that 00:33:07.360 --> 00:33:09.160 have been done in the last few years, 00:33:09.160 --> 00:33:10.660 to start investigating this. 00:33:10.660 --> 00:33:15.160 So this just shows, here, again, we have our lipid 2. 00:33:15.160 --> 00:33:16.930 We have our growing chain. 00:33:16.930 --> 00:33:18.740 And here we have our pentaglycine. 00:33:18.740 --> 00:33:21.550 So this is Staph. aureus. 00:33:21.550 --> 00:33:27.760 And we take D-alinine D-alinine, and form a cross-link 00:33:27.760 --> 00:33:29.740 and kick out D-alinine. 00:33:29.740 --> 00:33:31.720 And many of you have probably seen this before. 00:33:31.720 --> 00:33:34.070 I used to teach this in high school. 00:33:34.070 --> 00:33:36.820 [LAUGH] So that D-alinine D-alinine 00:33:36.820 --> 00:33:38.710 looks like penicillin. 00:33:38.710 --> 00:33:41.140 And we understand that this works-- 00:33:41.140 --> 00:33:43.580 it looks amazing, like a serine protease, which 00:33:43.580 --> 00:33:44.920 you're all very familiar with. 00:33:44.920 --> 00:33:47.140 We've seen this hundreds of times, now, 00:33:47.140 --> 00:33:48.250 in the earlier part-- 00:33:48.250 --> 00:33:49.900 to form this cross-link. 00:33:49.900 --> 00:33:53.890 And that cross-link is essential for the viability 00:33:53.890 --> 00:33:56.590 of the organism, in different ways. 00:33:56.590 --> 00:33:59.800 And you can imagine, if a bacteria is dividing, 00:33:59.800 --> 00:34:02.620 that you might have different peptidoglycal structure 00:34:02.620 --> 00:34:06.010 at the site, where the two dividing bacteria are 00:34:06.010 --> 00:34:07.390 going to split apart. 00:34:07.390 --> 00:34:08.949 So that might be why you want to have 00:34:08.949 --> 00:34:14.210 multiple glycosyltransferases in this overall process. 00:34:14.210 --> 00:34:14.710 OK. 00:34:14.710 --> 00:34:19.909 So this is just a cartoon that shows you targets. 00:34:19.909 --> 00:34:22.219 These are all natural products. 00:34:22.219 --> 00:34:23.710 Here's penicillin. 00:34:23.710 --> 00:34:24.460 It targets-- 00:34:24.460 --> 00:34:26.830 It looks-- not in this picture, but you 00:34:26.830 --> 00:34:28.030 can use your imagination. 00:34:28.030 --> 00:34:31.360 It looks just like D-alinine D-alinine. 00:34:31.360 --> 00:34:34.239 Binds in the active site, and covalently 00:34:34.239 --> 00:34:37.570 modifies a serine involved in that reaction. 00:34:37.570 --> 00:34:38.440 Moenomycin. 00:34:38.440 --> 00:34:40.150 What does this look like? 00:34:40.150 --> 00:34:41.260 This is sort of amazing. 00:34:41.260 --> 00:34:43.600 It's got this lipid thing, hanging off the end. 00:34:43.600 --> 00:34:45.159 That's a natural product. 00:34:45.159 --> 00:34:49.480 It binds, also, to the glycosyltransferase. 00:34:49.480 --> 00:34:51.730 And people are actively investigating this. 00:34:51.730 --> 00:34:54.340 You can imagine, this is not so easy to make 00:34:54.340 --> 00:34:57.030 as a new antibiotic. 00:34:57.030 --> 00:35:00.010 And then we have vancomycin, and vancomycin 00:35:00.010 --> 00:35:02.340 is able to bind D-alinine D-alinine. 00:35:02.340 --> 00:35:06.160 So these are all natural products that target cell wall. 00:35:06.160 --> 00:35:09.450 And, by far and away, the penicillins 00:35:09.450 --> 00:35:11.840 are the ones that are used much more prevalently. 00:35:11.840 --> 00:35:13.810 We have hundreds of variations of the theme. 00:35:13.810 --> 00:35:17.680 And, again, it's the war between the bacteria and the human, 00:35:17.680 --> 00:35:20.860 to figure out how to keep themselves growing. 00:35:20.860 --> 00:35:24.190 And so we have many variations on the beta-lactams. 00:35:24.190 --> 00:35:27.420 And you can take this even a step further, if you go-- 00:35:27.420 --> 00:35:30.550 in addition to the peptidoglycan, 00:35:30.550 --> 00:35:34.030 you have polymers of teichoic acid-- 00:35:34.030 --> 00:35:35.680 which I'm not [LAUGH] going to go into. 00:35:35.680 --> 00:35:38.820 But now people, for the first time, this year, 00:35:38.820 --> 00:35:42.040 have been able to reconstitute this polymer biosynthetic 00:35:42.040 --> 00:35:42.610 pathway. 00:35:42.610 --> 00:35:46.570 And this is a new target for design of the antibacterial. 00:35:46.570 --> 00:35:49.540 So I think it's exciting times, and we have really smart people 00:35:49.540 --> 00:35:51.520 working on this problem. 00:35:51.520 --> 00:35:54.310 And they now, for the first time, can set up the assays, 00:35:54.310 --> 00:35:56.350 so they can screen for small molecules that 00:35:56.350 --> 00:36:00.400 hopefully can target cell wall, which is unique to bacteria. 00:36:00.400 --> 00:36:01.270 OK. 00:36:01.270 --> 00:36:04.540 So what I want to do is talk about, 00:36:04.540 --> 00:36:06.100 in the last few minutes, as we're now 00:36:06.100 --> 00:36:07.460 moving into Staph. aureus. 00:36:07.460 --> 00:36:07.960 OK? 00:36:07.960 --> 00:36:10.690 And we're going to focus in on heme uptake rather 00:36:10.690 --> 00:36:12.130 than siderophore uptake. 00:36:12.130 --> 00:36:15.940 But if you look at this, what do we know about Staph. aureus? 00:36:15.940 --> 00:36:20.080 We know what a bit, because everybody and his brother 00:36:20.080 --> 00:36:22.810 has been studying it because of the problems with resistance. 00:36:22.810 --> 00:36:28.210 So, here, again, Staph. aureus actually 00:36:28.210 --> 00:36:34.190 has two biosynthetic pathways encoded in its genome. 00:36:34.190 --> 00:36:39.590 And what these pathways code for are these two siderophores. 00:36:39.590 --> 00:36:40.090 OK? 00:36:40.090 --> 00:36:42.830 And if you look at this, what's unusual? 00:36:42.830 --> 00:36:44.440 Does anybody see anything unusual 00:36:44.440 --> 00:36:47.680 about the siderophore structure, if you look at it carefully? 00:36:51.340 --> 00:36:53.550 I don't want to spend a lot of time on this, 00:36:53.550 --> 00:36:55.690 but what do you see in the structure? 00:36:55.690 --> 00:36:56.600 Can you read it? 00:36:56.600 --> 00:36:59.090 Or, if you brought your handout, you can probably read it. 00:36:59.090 --> 00:37:01.370 Since I insist on having the windows open, 00:37:01.370 --> 00:37:02.610 it's harder to read this. 00:37:02.610 --> 00:37:06.800 But what do we see, in siderophore, 00:37:06.800 --> 00:37:11.840 in this siderophore, Staphyloferrin A? 00:37:11.840 --> 00:37:13.960 See anything you recognize? 00:37:13.960 --> 00:37:14.460 Yeah. 00:37:14.460 --> 00:37:15.260 AUDIENCE: Some citrates? 00:37:15.260 --> 00:37:16.510 JOANNE STUBBE: Yeah, citrates. 00:37:16.510 --> 00:37:18.410 So, again, we're using citrate. 00:37:18.410 --> 00:37:22.400 We saw polycitrate can bind iron as a siderophore in itself. 00:37:22.400 --> 00:37:24.170 And, in fact, most gram-negative bacteria 00:37:24.170 --> 00:37:27.310 have iron-siderophore uptake system. 00:37:27.310 --> 00:37:29.280 Here, actually, all of these-- 00:37:29.280 --> 00:37:32.720 if you look at this carefully, the biosynthetic pathway, 00:37:32.720 --> 00:37:36.060 you know, is made out of basic metabolites. 00:37:36.060 --> 00:37:36.560 OK? 00:37:36.560 --> 00:37:40.190 That you see out of normal, central metabolic pathways. 00:37:40.190 --> 00:37:46.430 And what happens is, there's an ABC transporter and an ATPase-- 00:37:46.430 --> 00:37:48.270 FhuC is an ATPase-- 00:37:48.270 --> 00:37:50.900 all of this is written down in your notes-- 00:37:50.900 --> 00:37:57.080 that allow the siderophore to bring iron into the cell. 00:37:57.080 --> 00:37:58.580 And I think what's interesting here, 00:37:58.580 --> 00:38:00.500 and I've already pointed this out, 00:38:00.500 --> 00:38:05.880 in addition to the siderophores that the organism makes it also 00:38:05.880 --> 00:38:12.750 has a generic transporter that allows siderophores 00:38:12.750 --> 00:38:16.000 made by other organisms to bring iron into the cell. 00:38:16.000 --> 00:38:18.900 And so, again, that's a strategy that's 00:38:18.900 --> 00:38:20.380 used over and over again. 00:38:20.380 --> 00:38:25.410 So here's a xenosiderophore transport system, 00:38:25.410 --> 00:38:27.070 desperately trying to get iron. 00:38:27.070 --> 00:38:27.690 OK. 00:38:27.690 --> 00:38:30.690 So the ones we're going to be talking about and focusing on 00:38:30.690 --> 00:38:34.158 specifically are the heme uptake systems. 00:38:34.158 --> 00:38:35.700 And these are the ones you've already 00:38:35.700 --> 00:38:39.180 hopefully thought about, now, from your problem set. 00:38:39.180 --> 00:38:41.040 We have to extract-- 00:38:41.040 --> 00:38:45.670 I just told you that red blood cells have most of the iron. 00:38:45.670 --> 00:38:47.010 So Staph. 00:38:47.010 --> 00:38:50.820 has been incredibly creative in generating endotoxins 00:38:50.820 --> 00:38:54.870 that lyse red blood cells, allowing the heme-- 00:38:54.870 --> 00:38:56.640 hemoglobin, OK? 00:38:56.640 --> 00:39:07.320 So we have endotoxins from the organism 00:39:07.320 --> 00:39:12.070 that lyse red blood cells. 00:39:12.070 --> 00:39:16.330 And so what you get out, then, is hemoglobin. 00:39:16.330 --> 00:39:19.960 Which, again, has four hemes and iron. 00:39:19.960 --> 00:39:21.250 And you want to get-- 00:39:21.250 --> 00:39:23.500 the key thing is to get the iron out of the heme. 00:39:23.500 --> 00:39:27.220 So you want to be able to extract the iron out 00:39:27.220 --> 00:39:27.820 of the heme. 00:39:27.820 --> 00:39:32.255 And also-- and I have this down in your nomenclature-- 00:39:32.255 --> 00:39:34.630 it turns out red blood cells have another protein, called 00:39:34.630 --> 00:39:37.150 "haptoglobin," that binds to hemoglobin. 00:39:37.150 --> 00:39:39.820 And that's another place that these organisms have 00:39:39.820 --> 00:39:42.190 evolved to extract the heme-- 00:39:42.190 --> 00:39:44.170 to extract the heme. 00:39:44.170 --> 00:39:45.670 So, in all of these cases, you're 00:39:45.670 --> 00:39:52.270 extracting the heme out of the protein. 00:39:52.270 --> 00:39:57.760 And so, over here, you see the two different ways to do that. 00:39:57.760 --> 00:40:01.040 And we have different proteins that are able to do this. 00:40:01.040 --> 00:40:04.210 And then, eventually, the heme that's extracted 00:40:04.210 --> 00:40:06.970 is passed through this peptidoglycan, 00:40:06.970 --> 00:40:11.440 eventually to the plasma membrane, where 00:40:11.440 --> 00:40:13.510 the heme goes into the cytosol. 00:40:13.510 --> 00:40:15.590 And in this organism, to get it out, 00:40:15.590 --> 00:40:17.110 you have to break down the heme. 00:40:17.110 --> 00:40:21.730 You have to cleave it into pieces by the enzyme called 00:40:21.730 --> 00:40:23.340 "heme oxygenases." 00:40:23.340 --> 00:40:24.280 OK. 00:40:24.280 --> 00:40:26.830 So I don't want to really say very much 00:40:26.830 --> 00:40:32.290 about the siderophores, except to say-- 00:40:32.290 --> 00:40:35.950 let me comment on iron sensing. 00:40:35.950 --> 00:40:40.570 And you saw-- and this would be Staph. aureus, 00:40:40.570 --> 00:40:45.640 but it's in true iron-sensing for most bacteria. 00:40:45.640 --> 00:40:47.950 You saw iron-sensing predominantly 00:40:47.950 --> 00:40:49.450 at the translational level. 00:40:49.450 --> 00:40:50.430 Which was unusual. 00:40:50.430 --> 00:40:52.450 That's why we talked about it, in humans. 00:40:52.450 --> 00:40:55.330 Here, iron-sensing is predominantly 00:40:55.330 --> 00:40:57.130 at the transcriptional level. 00:40:57.130 --> 00:41:00.770 So this sensing occurs transcriptionally. 00:41:05.870 --> 00:41:12.640 And so you have a transcription factor, which is called "Fur." 00:41:12.640 --> 00:41:14.350 And Fur is a transcription factor. 00:41:14.350 --> 00:41:18.400 That name is used for almost all organisms. 00:41:18.400 --> 00:41:21.760 And I'm not going to say much about this, 00:41:21.760 --> 00:41:25.460 but we're going to look at the operon in a minute. 00:41:25.460 --> 00:41:28.840 But here's Fur. 00:41:28.840 --> 00:41:36.870 And if Fur has iron bound, what it does is a repressor. 00:41:36.870 --> 00:41:42.340 And it shuts down transcription of all the proteins 00:41:42.340 --> 00:41:44.560 that you might think it would shut down. 00:41:44.560 --> 00:41:47.920 They can no longer take up iron into the cell, 00:41:47.920 --> 00:41:50.420 because you have excess iron and you don't need anymore. 00:41:50.420 --> 00:41:52.900 Again, you want to control iron, because you 00:41:52.900 --> 00:41:55.210 have problems if you have too much iron 00:41:55.210 --> 00:41:57.130 with oxidative stress. 00:41:57.130 --> 00:41:57.630 OK. 00:41:57.630 --> 00:42:00.530 So, if you look at the operon-- 00:42:00.530 --> 00:42:01.030 let's see. 00:42:01.030 --> 00:42:04.650 So look at the operon, here. 00:42:04.650 --> 00:42:06.110 So here's the operon. 00:42:06.110 --> 00:42:08.860 And we're going to see that the key proteins involved 00:42:08.860 --> 00:42:13.010 in heme uptake are called the "Isd" proteins. 00:42:13.010 --> 00:42:18.470 And so, if you look at all of these Isd proteins, this Isd 00:42:18.470 --> 00:42:22.310 protein and that one, they all have these little Fur boxes. 00:42:22.310 --> 00:42:25.370 [LAUGH] So we have a Fur box ahead, 00:42:25.370 --> 00:42:28.700 which regulates whether you're going to make a siderophore 00:42:28.700 --> 00:42:31.640 or whether you're going to make all this equipment required 00:42:31.640 --> 00:42:32.780 to take up heme. 00:42:32.780 --> 00:42:35.420 So all of that makes sense, and people 00:42:35.420 --> 00:42:40.310 have studied this extensively, in many of these organisms. 00:42:40.310 --> 00:42:40.970 OK. 00:42:40.970 --> 00:42:45.770 So what I want to do now is, I'm going to show you this cartoon 00:42:45.770 --> 00:42:46.770 overview. 00:42:46.770 --> 00:42:49.790 And then we'll look at a few experiments 00:42:49.790 --> 00:42:56.030 that people have done to try to look at what basis in reality 00:42:56.030 --> 00:42:58.850 this cartoon model has to what actually happens 00:42:58.850 --> 00:43:00.200 inside the cell. 00:43:00.200 --> 00:43:01.632 So let's look at-- 00:43:01.632 --> 00:43:03.590 I can never remember the names of these things. 00:43:03.590 --> 00:43:07.670 I'm just going to call it the "Isd proteins." 00:43:07.670 --> 00:43:10.340 And so there are two proteins, we're going to see, 00:43:10.340 --> 00:43:14.540 that are closest to the surface, that directly interact with 00:43:14.540 --> 00:43:17.180 hemoglobin-- or haptoglobin and hemoglobin-- 00:43:17.180 --> 00:43:19.400 the other ones that are going to somehow get 00:43:19.400 --> 00:43:22.700 the heme out of the proteins. 00:43:22.700 --> 00:43:27.100 And then these each have little NEAT domains. 00:43:27.100 --> 00:43:29.630 So N1 is a NEAT domain. 00:43:29.630 --> 00:43:32.840 So they have a name for that, which I've also written down. 00:43:32.840 --> 00:43:35.120 It's, like, 120 amino acids. 00:43:35.120 --> 00:43:37.880 And each one of these proteins sometimes has two, 00:43:37.880 --> 00:43:40.100 sometimes has three, sometimes has one, 00:43:40.100 --> 00:43:42.480 and they're structurally all the same. 00:43:42.480 --> 00:43:46.940 But it turns out that you can't just pick up one and replace it 00:43:46.940 --> 00:43:47.900 with another. 00:43:47.900 --> 00:43:49.400 There's something about the spinach 00:43:49.400 --> 00:43:51.860 on each side of these NEAT domains 00:43:51.860 --> 00:43:56.600 that is key, you can imagine, for the directionality 00:43:56.600 --> 00:43:57.740 of the transfer. 00:43:57.740 --> 00:44:02.690 So you want something that the heme is going to get down here. 00:44:02.690 --> 00:44:04.790 You don't want something where the equilibrium 00:44:04.790 --> 00:44:06.470 is going to stay up there. 00:44:06.470 --> 00:44:08.610 So this is not an easy problem. 00:44:08.610 --> 00:44:12.650 And this is a problem that we discussed in the beginning-- 00:44:12.650 --> 00:44:15.770 the importance of exchange ligands. 00:44:15.770 --> 00:44:18.560 Because somehow we're going to have a heme in a little NEAT 00:44:18.560 --> 00:44:21.500 domain, but it's going to move into the next domain. 00:44:21.500 --> 00:44:22.850 It just doesn't hop. 00:44:22.850 --> 00:44:24.620 It's covalently bound. 00:44:24.620 --> 00:44:28.490 So how do you transfer one heme to the next heme? 00:44:28.490 --> 00:44:30.470 And we have a lot of structural information, 00:44:30.470 --> 00:44:32.900 but I would say we still don't understand how 00:44:32.900 --> 00:44:35.180 these transfers actually occur. 00:44:35.180 --> 00:44:35.810 OK. 00:44:35.810 --> 00:44:38.477 So there's a couple other things that I want to point out, here. 00:44:38.477 --> 00:44:44.600 So IsB and IsH extract from heme and hemoglobin. 00:44:44.600 --> 00:44:47.000 This gives you a feeling, which you also 00:44:47.000 --> 00:44:50.540 saw from the problem set, that these little domains-- 00:44:50.540 --> 00:44:53.060 N1 domains, N2 domains-- 00:44:53.060 --> 00:44:54.320 are all NEAT domains. 00:44:54.320 --> 00:44:56.798 So we have multiple domains. 00:44:56.798 --> 00:44:58.340 And what we're going to see, and this 00:44:58.340 --> 00:45:01.790 is key to the way these organisms function, 00:45:01.790 --> 00:45:05.750 is that these Isd proteins are covalently 00:45:05.750 --> 00:45:07.790 attached to the peptidoglycan. 00:45:07.790 --> 00:45:21.630 So the issue is, we need to covalently attach the Isd 00:45:21.630 --> 00:45:28.800 proteins to the peptidoglycans. 00:45:28.800 --> 00:45:31.890 And the protein-- 00:45:31.890 --> 00:45:35.280 There are two different proteins that do this. 00:45:35.280 --> 00:45:43.410 So the Isd proteins have ZIP codes. 00:45:43.410 --> 00:45:44.520 Where have we seen this? 00:45:44.520 --> 00:45:48.060 We see this over and over and over again. 00:45:48.060 --> 00:45:50.820 We have little sequences of peptides that are 00:45:50.820 --> 00:45:53.220 recognized by another protein. 00:45:53.220 --> 00:45:54.380 OK? 00:45:54.380 --> 00:45:55.560 So we have ZIP codes. 00:45:55.560 --> 00:45:57.990 And the ZIP codes, I'll just say "see PowerPoint" 00:45:57.990 --> 00:46:00.420 for the sequence. 00:46:00.420 --> 00:46:04.110 And it turns out, if you look over here, 00:46:04.110 --> 00:46:07.530 all of these proteins with a yellow anchor 00:46:07.530 --> 00:46:08.950 have little ZIP codes in them. 00:46:08.950 --> 00:46:09.450 OK? 00:46:09.450 --> 00:46:11.760 [LAUGH] And they're recognized by a protein 00:46:11.760 --> 00:46:13.560 called "sortase A." 00:46:13.560 --> 00:46:14.060 OK. 00:46:14.060 --> 00:46:18.712 So we'll see that, in addition to the Ist proteins, 00:46:18.712 --> 00:46:19.420 we have sortases. 00:46:22.590 --> 00:46:26.760 And we have sortase A and B, and they 00:46:26.760 --> 00:46:35.360 recognize the ZIP codes, distinct ZIP codes, 00:46:35.360 --> 00:46:39.540 and are required to attach the Isd proteins covalently 00:46:39.540 --> 00:46:41.010 to the peptidoglycan. 00:46:41.010 --> 00:46:47.970 And in the peptidoglycan of any gram-positive, a lot of things 00:46:47.970 --> 00:46:51.270 are covalently attached to the peptidoglycan. 00:46:51.270 --> 00:46:52.890 So, I mean, can you imagine-- how 00:46:52.890 --> 00:46:56.790 dense do you need these proteins, to be 00:46:56.790 --> 00:46:58.110 able to do these switches? 00:46:58.110 --> 00:47:00.270 I mean, this is a cartoon overview 00:47:00.270 --> 00:47:02.850 that really doesn't tell you anything 00:47:02.850 --> 00:47:04.320 about the complexity of all that-- 00:47:04.320 --> 00:47:06.190 what does a peptidoglycan look like? 00:47:06.190 --> 00:47:09.780 Well, it's got a lot of water and a lot of space 00:47:09.780 --> 00:47:12.100 in between these N-acetylglucosamine, 00:47:12.100 --> 00:47:14.410 N-acetylmuramic acids. 00:47:14.410 --> 00:47:18.080 So this is involved in the covalent attachment. 00:47:21.920 --> 00:47:25.640 And it, in fact, involves what you've seen over 00:47:25.640 --> 00:47:31.770 and over again-- involves covalent catalysis 00:47:31.770 --> 00:47:35.160 with a cystine in its active site. 00:47:35.160 --> 00:47:36.240 OK? 00:47:36.240 --> 00:47:41.670 So what I want to do is briefly look at what 00:47:41.670 --> 00:47:43.325 these sortases actually do. 00:47:43.325 --> 00:47:44.950 I'm not going to write it on the board. 00:47:44.950 --> 00:47:48.755 I'll walk you through it and then, next time-- 00:47:48.755 --> 00:47:50.130 hopefully, you've already thought 00:47:50.130 --> 00:47:51.840 about this in some form, but I'll walk you through it 00:47:51.840 --> 00:47:53.462 and go through it next time. 00:47:53.462 --> 00:47:55.170 And then what we're going to do is simply 00:47:55.170 --> 00:47:58.260 look at a few experiments with Isd proteins, 00:47:58.260 --> 00:48:01.740 to look at this movement of heme across the membrane, 00:48:01.740 --> 00:48:03.870 similar to the kinds of experiments 00:48:03.870 --> 00:48:08.700 that you had on the problem set that was due this week. 00:48:08.700 --> 00:48:09.230 OK. 00:48:09.230 --> 00:48:11.280 So, because I don't have much time 00:48:11.280 --> 00:48:13.968 and I can't write that fast and you can't write that fast, 00:48:13.968 --> 00:48:15.510 either, [LAUGH] I'm going to walk you 00:48:15.510 --> 00:48:18.150 through sort of what's going on in this reaction. 00:48:18.150 --> 00:48:18.870 OK. 00:48:18.870 --> 00:48:21.360 So, remember, all of these things 00:48:21.360 --> 00:48:24.370 are anchored to the plasma membrane. 00:48:24.370 --> 00:48:25.620 OK, so that's the other thing. 00:48:25.620 --> 00:48:28.740 Sometimes they have single, transmembrane-spanning regions. 00:48:28.740 --> 00:48:31.560 Sometimes they have lipids that are actually bound. 00:48:31.560 --> 00:48:33.850 I wanted to say one other thing, here. 00:48:33.850 --> 00:48:36.750 So these yellow things are anchored 00:48:36.750 --> 00:48:41.100 by sortase A. The blue thing is anchored by sortase B. 00:48:41.100 --> 00:48:45.870 And IsdE is anchored by a lipid, covalently bound. 00:48:45.870 --> 00:48:48.990 OK, so we have three different strategies, to anchor. 00:48:48.990 --> 00:48:50.220 OK? 00:48:50.220 --> 00:48:52.630 And every organism is distinct. 00:48:52.630 --> 00:48:55.200 Whoops, I'm going the wrong way. 00:48:55.200 --> 00:48:57.550 OK, so what happens in this reaction? 00:48:57.550 --> 00:49:00.170 So here's our ZIP code. 00:49:00.170 --> 00:49:03.810 OK, and what we know about this-- and here's sortase A. 00:49:03.810 --> 00:49:08.030 Sortase A is anchored to the plasma membrane. 00:49:08.030 --> 00:49:11.060 In a further cartoon, they don't have it anchored, 00:49:11.060 --> 00:49:13.610 but I tell you it's anchored. 00:49:13.610 --> 00:49:17.960 And we know we get cleavage between threonine and glycine. 00:49:17.960 --> 00:49:20.450 And we know we have a sulfhydryl on the active site. 00:49:20.450 --> 00:49:24.290 So, this chemistry, we've seen over and over and over and over 00:49:24.290 --> 00:49:26.840 again, whether it's with serine or with a cystine, 00:49:26.840 --> 00:49:28.640 you have to have the right equipment 00:49:28.640 --> 00:49:30.930 to acylate the enzyme. 00:49:30.930 --> 00:49:34.700 So what happens here is you acylate the enzyme. 00:49:34.700 --> 00:49:38.120 And so this is the part of the protein that's 00:49:38.120 --> 00:49:42.240 going to get transferred, ultimately, to-- this 00:49:42.240 --> 00:49:45.390 is lipid 2, with the pentaglycine. 00:49:45.390 --> 00:49:47.100 And at the end of the pentaglycine 00:49:47.100 --> 00:49:48.810 you have an amino group. 00:49:48.810 --> 00:49:51.600 That's-- you're going to attach this protein, 00:49:51.600 --> 00:49:57.650 IsdA or IsdB to this lyse-- 00:49:57.650 --> 00:50:05.520 the end-terminal amino group of glycine in the pentaglycine. 00:50:05.520 --> 00:50:06.900 So you form. 00:50:06.900 --> 00:50:11.040 Again, you cleave this peptide bond. 00:50:11.040 --> 00:50:15.630 And you have this piece left over from your Isd protein. 00:50:15.630 --> 00:50:18.900 You now have this covalently attached to the sortase. 00:50:18.900 --> 00:50:21.000 And again, what you're doing is going 00:50:21.000 --> 00:50:23.280 to regenerate the sortase, so you 00:50:23.280 --> 00:50:25.330 can do more of these reactions. 00:50:25.330 --> 00:50:31.640 And here you're forming your linkage to the Isd protein. 00:50:31.640 --> 00:50:32.140 OK? 00:50:32.140 --> 00:50:34.840 Does everybody see what's going on in that reaction? 00:50:34.840 --> 00:50:38.760 So another cartoon version of this, and then I'll stop here. 00:50:38.760 --> 00:50:40.290 This is a more chemical version. 00:50:40.290 --> 00:50:43.560 Again, this is the sortase. 00:50:43.560 --> 00:50:45.320 Here is your amino-acid sequence. 00:50:45.320 --> 00:50:47.070 You go through a tetrahedral intermediate. 00:50:47.070 --> 00:50:49.545 This is all a figment of our imaginations, 00:50:49.545 --> 00:50:53.250 [LAUGH] based on what we think-- what we do understand, 00:50:53.250 --> 00:50:56.880 in the test tube of peptide bond hydrolysis-- 00:50:56.880 --> 00:50:59.220 not so much in the enzymes. 00:50:59.220 --> 00:51:04.530 But you generate an acylated attached protein. 00:51:04.530 --> 00:51:06.780 And then we have our pentaglycine, 00:51:06.780 --> 00:51:09.960 the terminal amino group that goes through, 00:51:09.960 --> 00:51:12.840 again, a tetrahedral intermediate to form 00:51:12.840 --> 00:51:14.820 this linkage. 00:51:14.820 --> 00:51:17.670 So what's happening-- I think this is, like, so, again, 00:51:17.670 --> 00:51:18.180 amazing-- 00:51:18.180 --> 00:51:21.720 what's happening is, you're transferring-- 00:51:21.720 --> 00:51:24.360 you've got your lipid 2, and you've 00:51:24.360 --> 00:51:27.450 transferred it across this membrane 00:51:27.450 --> 00:51:31.120 into the outside of your bacteria. 00:51:31.120 --> 00:51:32.370 So you've gotta hang it there. 00:51:32.370 --> 00:51:35.670 That's why you need these big, huge lipids. 00:51:35.670 --> 00:51:39.740 And what you're going to do is attach to this pentaglycine. 00:51:39.740 --> 00:51:45.540 You're going to attach each of these Isd proteins, covalently. 00:51:45.540 --> 00:51:46.850 And then what you do-- 00:51:46.850 --> 00:51:48.000 So you make this guy. 00:51:48.000 --> 00:51:52.090 Then you attach this whole thing onto the growing polypeptide 00:51:52.090 --> 00:51:52.590 chain. 00:51:52.590 --> 00:51:55.020 I mean, this is, like, an amazing machine 00:51:55.020 --> 00:51:57.900 that they've unraveled, I think, from studies 00:51:57.900 --> 00:52:01.600 that have been done in the last five years or so. 00:52:01.600 --> 00:52:04.650 So, next time, we'll come back and talk a little bit 00:52:04.650 --> 00:52:06.960 about the Isd proteins, but I think 00:52:06.960 --> 00:52:08.200 you should be fine, looking. 00:52:08.200 --> 00:52:10.575 You've looked at-- all you're doing is transferring heme, 00:52:10.575 --> 00:52:12.660 and we don't understand the detailed mechanism 00:52:12.660 --> 00:52:13.890 of how that happens. 00:52:13.890 --> 00:52:17.510 That's something hopefully some of you will figure out.