WEBVTT 00:00:00.500 --> 00:00:02.810 The following content is provided under a Creative 00:00:02.810 --> 00:00:04.380 Commons license. 00:00:04.380 --> 00:00:06.670 Your support will help MIT OpenCourseWare 00:00:06.670 --> 00:00:11.010 continue to offer high-quality educational resources for free. 00:00:11.010 --> 00:00:13.670 To make a donation or view additional materials 00:00:13.670 --> 00:00:17.600 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:17.600 --> 00:00:18.480 at ocw.mit.edu. 00:00:25.847 --> 00:00:27.430 PROFESSOR: We're going to get started. 00:00:27.430 --> 00:00:31.390 And today we're going to move forward with the protein 00:00:31.390 --> 00:00:35.410 degradation module, and looking at the degradation 00:00:35.410 --> 00:00:37.900 chamber of E. coli, ClpXP. 00:00:37.900 --> 00:00:41.296 So this is a degradation machine. 00:00:41.296 --> 00:00:45.610 And effectively, what we see in this case, 00:00:45.610 --> 00:00:47.800 and as mentioned in lecture last time, 00:00:47.800 --> 00:00:51.370 is that there are gigantic chambers that 00:00:51.370 --> 00:00:54.460 isolate protease active sites. 00:00:54.460 --> 00:00:57.040 And so we're going to examine this particular machinery 00:00:57.040 --> 00:00:59.320 as a paradigm here. 00:00:59.320 --> 00:01:02.710 So the Clp system was first identified in E. coli, 00:01:02.710 --> 00:01:05.373 and it's highly conserved. 00:01:05.373 --> 00:01:06.790 And what we'll see is that there's 00:01:06.790 --> 00:01:11.140 encapsulation of an active site in a large degradation chamber. 00:01:11.140 --> 00:01:14.110 So there's two components, ClpX and ClpP. 00:01:14.110 --> 00:01:16.360 And so we're going to look at both of these components 00:01:16.360 --> 00:01:19.960 individually, and then see how this machine works. 00:01:19.960 --> 00:01:26.560 And so ClpP is the proteasome. 00:01:32.260 --> 00:01:33.420 And it's a serine protease. 00:01:36.520 --> 00:01:39.940 So we talked about serine proteases last time. 00:01:39.940 --> 00:01:44.910 So there's the catalytic triad of serine, histidine, 00:01:44.910 --> 00:01:46.120 and aspartate. 00:01:46.120 --> 00:01:48.790 So there's formation of a covalent 00:01:48.790 --> 00:01:50.140 acyl-enzyme intermediate. 00:01:50.140 --> 00:01:52.390 We learned that the serine residue is the active site 00:01:52.390 --> 00:01:53.680 nucleophile. 00:01:53.680 --> 00:01:57.070 And what we're going to see is that this degradation chamber 00:01:57.070 --> 00:01:58.540 has 14 active sites. 00:02:07.640 --> 00:02:17.900 And what this does is degrade proteins 00:02:17.900 --> 00:02:21.290 into short polypeptides. 00:02:21.290 --> 00:02:28.970 So those are peptides of about seven to eight amino acids. 00:02:33.300 --> 00:02:37.800 And so if we consider a cartoon of the structure, what we find 00:02:37.800 --> 00:02:46.720 is that we have two back-to-back rings here. 00:02:46.720 --> 00:02:49.390 And each of these rings is a 7-mer. 00:02:49.390 --> 00:02:50.650 So we have two heptamers. 00:02:57.150 --> 00:03:01.470 In terms of size, they're approximately 90 angstroms 00:03:01.470 --> 00:03:08.220 here and approximately 90 angstroms across. 00:03:08.220 --> 00:03:10.260 And then this region between the two rings 00:03:10.260 --> 00:03:12.015 is sometimes referred to as the equator. 00:03:17.030 --> 00:03:18.860 So here we have two back-to-back rings. 00:03:26.410 --> 00:03:28.195 And these rings generate a chamber. 00:03:36.350 --> 00:03:44.060 And proteins upwards of about 70 kilodaltons can fit. 00:03:51.180 --> 00:03:54.100 And so if we take this, looking at from the side, 00:03:54.100 --> 00:03:56.400 and just rotate 90 degrees to ask, what does it 00:03:56.400 --> 00:03:59.250 look like from the top? 00:03:59.250 --> 00:04:00.450 So this is a side view. 00:04:25.880 --> 00:04:26.380 OK. 00:04:26.380 --> 00:04:32.320 What we see is that there is a very small pore here. 00:04:32.320 --> 00:04:34.540 So we have the seven subunits. 00:04:34.540 --> 00:04:38.590 And then in the center there is an axial pore. 00:04:42.160 --> 00:04:46.445 And this pore is small, about 10 angstroms in diameter. 00:04:55.240 --> 00:04:56.890 So when thinking about that size, 00:04:56.890 --> 00:05:00.160 we need to think about the size of some large protein, right? 00:05:00.160 --> 00:05:03.190 If we have something on the order of 70 kilodaltons 00:05:03.190 --> 00:05:05.710 with a fold, that protein's not going 00:05:05.710 --> 00:05:09.430 to fit through this hole in that state here. 00:05:09.430 --> 00:05:10.600 OK? 00:05:10.600 --> 00:05:13.720 So it's too small for a big, folded protein. 00:05:13.720 --> 00:05:17.440 But then basically, if we take this and, rather than just 00:05:17.440 --> 00:05:19.300 looking at the top, we cut through 00:05:19.300 --> 00:05:22.390 and ask what's going on in the interior, what do we see? 00:05:25.840 --> 00:05:27.220 OK, so cut through. 00:05:39.550 --> 00:05:44.200 What we see now is that there's a chamber 00:05:44.200 --> 00:05:45.790 of about 51 angstroms. 00:05:49.030 --> 00:05:55.000 OK, so this is the interior degradation chamber. 00:06:02.850 --> 00:06:05.640 OK, so one question we're going to address as we move forward 00:06:05.640 --> 00:06:08.490 is that, how is it that a polypeptide gets 00:06:08.490 --> 00:06:11.370 through this hole into the degradation chamber that 00:06:11.370 --> 00:06:15.870 can accommodate a protein up to 17 kilodaltons? 00:06:15.870 --> 00:06:22.080 So small axial pore versus large degradation chamber here. 00:06:24.660 --> 00:06:30.630 So we'll look at some structures of ClpP and then go on to ClpX. 00:06:30.630 --> 00:06:34.500 So what we're looking at here are effectively 00:06:34.500 --> 00:06:37.350 what I've drawn out in cartoon form on the board. 00:06:37.350 --> 00:06:40.650 So here we have the side view of ClpP. 00:06:40.650 --> 00:06:44.100 We have the top ring, the bottom ring, and here's 00:06:44.100 --> 00:06:46.650 the region between the two, the equator. 00:06:46.650 --> 00:06:48.780 If we look at the top, here they're 00:06:48.780 --> 00:06:53.010 describing the axial pore as a portal. 00:06:53.010 --> 00:06:54.360 And here's the cutaway view. 00:06:54.360 --> 00:06:56.250 So this hole is very small. 00:06:56.250 --> 00:06:58.710 And if we look at the cutaway view, what we see 00:06:58.710 --> 00:07:02.130 is the degradation chamber here. 00:07:02.130 --> 00:07:04.740 And basically, the seven different serine 00:07:04.740 --> 00:07:09.510 protease active sites are shown here. 00:07:09.510 --> 00:07:13.320 And this is a side view cutting through the middle here. 00:07:13.320 --> 00:07:17.490 So we see that these serine protease active sites are 00:07:17.490 --> 00:07:21.510 down in this region here. 00:07:21.510 --> 00:07:25.380 If we take another view and look at-- again, this is cutaway, 00:07:25.380 --> 00:07:28.500 so cut through the side view-- 00:07:28.500 --> 00:07:32.070 what we can look at here is the machinery in the active site. 00:07:32.070 --> 00:07:36.480 So we learned last time about the catalytic triad, 00:07:36.480 --> 00:07:39.330 with the aspartate, histidine, and serine. 00:07:39.330 --> 00:07:41.880 And in this particular structure there's 00:07:41.880 --> 00:07:46.530 a serine protease inhibitor bound to the serine side chain 00:07:46.530 --> 00:07:47.700 here. 00:07:47.700 --> 00:07:51.030 So that can serve machinery. 00:07:51.030 --> 00:07:54.600 If we look at the structure of an individual ClpP subunit, 00:07:54.600 --> 00:07:59.250 those are shown here from several different organisms. 00:07:59.250 --> 00:08:02.880 What we see-- if we can think about this as the top part, 00:08:02.880 --> 00:08:05.280 and this is the bottom part of one ring-- 00:08:05.280 --> 00:08:08.550 we see there's a region with axial loops. 00:08:08.550 --> 00:08:12.930 There's a head domain and what's called a handle region. 00:08:12.930 --> 00:08:16.710 And the catalytic triad is located at the juncture 00:08:16.710 --> 00:08:18.690 of the head and handle region. 00:08:18.690 --> 00:08:21.600 And you can, again, look back to the cutaway views 00:08:21.600 --> 00:08:24.690 to orient that within the whole chamber. 00:08:24.690 --> 00:08:28.020 These axial loops, we'll see, are important for interaction 00:08:28.020 --> 00:08:29.580 with ClpX. 00:08:29.580 --> 00:08:31.950 And we'll talk about that component 00:08:31.950 --> 00:08:34.630 of the machine in a moment. 00:08:34.630 --> 00:08:39.389 Here, again, just structures of ClpP from various organisms, 00:08:39.389 --> 00:08:42.510 E. coli, Streptococcus, human. 00:08:42.510 --> 00:08:47.620 We see that they're all very similar here. 00:08:47.620 --> 00:08:50.780 So what is ClpX? 00:08:50.780 --> 00:08:51.405 Moving forward. 00:09:01.740 --> 00:09:02.250 OK. 00:09:02.250 --> 00:09:06.510 So ClpX is effectively an accessory protein. 00:09:11.680 --> 00:09:13.480 And in some respects we can think about it 00:09:13.480 --> 00:09:15.370 as a lid to the proteasome. 00:09:21.580 --> 00:09:22.080 OK. 00:09:22.080 --> 00:09:22.705 It's a hexamer. 00:09:27.910 --> 00:09:31.570 So there's a mismatch here, in terms of the number of subunits 00:09:31.570 --> 00:09:32.870 in ClpP and ClpX. 00:09:32.870 --> 00:09:35.670 This is different from what we saw with GroEL, GroES, 00:09:35.670 --> 00:09:39.360 where they are both heptamers. 00:09:39.360 --> 00:09:41.100 ClpX is a hexamer. 00:09:41.100 --> 00:09:43.880 And it's an AAA-- 00:09:43.880 --> 00:09:46.700 so triple-A-plus unfoldase. 00:09:51.240 --> 00:09:56.630 It's an ATPase here. 00:09:56.630 --> 00:09:59.250 And effectively, what we'll see is 00:09:59.250 --> 00:10:03.810 that ClpX has an important role as an accessory protein that 00:10:03.810 --> 00:10:06.630 unfolds the polypeptide that's destined 00:10:06.630 --> 00:10:08.460 for degradation by ClpP. 00:10:19.560 --> 00:10:21.450 So it unfolds the polypeptide, and we're 00:10:21.450 --> 00:10:23.760 going to have to ask how as we go through. 00:10:32.700 --> 00:10:33.220 OK. 00:10:33.220 --> 00:10:35.880 And in addition to unfolding, it also 00:10:35.880 --> 00:10:39.180 threads that polypeptide that's being unfolded in 00:10:39.180 --> 00:10:40.890 through the axial pores such that it 00:10:40.890 --> 00:10:43.980 can reach the degradation chamber, 00:10:43.980 --> 00:10:56.235 and threads it into the degradation chamber. 00:11:02.500 --> 00:11:13.720 And so if we look at ClpX from a top view, 00:11:13.720 --> 00:11:14.780 again, we have a hole. 00:11:25.230 --> 00:11:33.120 And we have a 6-mer here. 00:11:33.120 --> 00:11:40.200 So if we take a look, in this particular depiction what 00:11:40.200 --> 00:11:44.680 we're seeing are the top views and the side views. 00:11:44.680 --> 00:11:46.560 So here we have ClpP. 00:11:46.560 --> 00:11:48.450 Here we have ClpX. 00:11:48.450 --> 00:11:53.190 And what we want to ask is, how is it that ClpX binds to ClpP? 00:11:53.190 --> 00:11:56.290 And how is this mismatch in terms of the number of subunits 00:11:56.290 --> 00:11:57.450 accommodated, right? 00:11:57.450 --> 00:11:59.700 So it's not that one subunit is precisely 00:11:59.700 --> 00:12:02.260 going to act with one, because we have six in-- 00:12:02.260 --> 00:12:02.760 throughout. 00:12:02.760 --> 00:12:06.720 Because we have six in ClpX and seven in ClpP. 00:12:06.720 --> 00:12:24.020 And so if we think about how ClpX binds to ClpP, what we see 00:12:24.020 --> 00:12:27.530 is that we have the 6-mer. 00:12:27.530 --> 00:12:29.180 So we're looking from the side view. 00:12:31.790 --> 00:12:35.615 And there's some loops that are called IGF loops. 00:12:38.840 --> 00:12:40.610 So these are tripeptide motifs. 00:12:43.990 --> 00:12:44.865 And they're flexible. 00:12:49.250 --> 00:12:54.740 And these loops interact with hydrophobic regions of ClpP. 00:13:11.330 --> 00:13:14.660 And this flexibility helps accommodate 00:13:14.660 --> 00:13:17.000 the six versus seven subunits. 00:13:17.000 --> 00:13:23.240 So if we look here, here we see these tripeptide loops. 00:13:23.240 --> 00:13:25.190 And see, we're only seeing three. 00:13:25.190 --> 00:13:30.350 But there's one per subunit, so 1, 2, 3, 4, 5, 6 here. 00:13:30.350 --> 00:13:33.020 And then what's shown here in red 00:13:33.020 --> 00:13:37.050 are the hydrophobic regions of ClpP where these can bind. 00:13:39.770 --> 00:13:44.060 And where do we see these regions on ClpP here? 00:13:44.060 --> 00:13:49.190 They're a bit removed from the axial pore here. 00:13:49.190 --> 00:13:52.700 So how many of these loops are needed? 00:13:52.700 --> 00:13:56.150 Just to note, there's been studies done where 00:13:56.150 --> 00:13:57.470 these residues are deleted. 00:13:57.470 --> 00:14:00.440 And the question is, how many of these motifs 00:14:00.440 --> 00:14:05.210 are important for this protein-protein interaction? 00:14:05.210 --> 00:14:07.890 And what's been found in test-tube studies 00:14:07.890 --> 00:14:11.150 is that a minimum of two are required to get interaction 00:14:11.150 --> 00:14:15.676 between ClpX and ClpP here. 00:14:15.676 --> 00:14:16.176 Yeah. 00:14:16.176 --> 00:14:20.960 AUDIENCE: Is it known how many actually interact in vivo? 00:14:20.960 --> 00:14:25.280 Like, do all six interact at any given time? 00:14:25.280 --> 00:14:28.080 PROFESSOR: I would presume so, but I don't know. 00:14:28.080 --> 00:14:28.580 Right? 00:14:28.580 --> 00:14:36.800 So we very much think of this as coming together as shown there. 00:14:36.800 --> 00:14:37.800 But don't know. 00:14:37.800 --> 00:14:38.820 Joanne, do you know? 00:14:38.820 --> 00:14:39.320 No. 00:14:39.320 --> 00:14:41.830 No. 00:14:41.830 --> 00:14:45.820 I would say it needs to be pretty stable. 00:14:45.820 --> 00:14:47.120 Like, there's always dynamics. 00:14:47.120 --> 00:14:50.290 But as we see how this machine works, 00:14:50.290 --> 00:14:53.380 this hole is going to have to allow the polypeptide to thread 00:14:53.380 --> 00:14:57.820 through and get through that axial pore for the polypeptide 00:14:57.820 --> 00:14:59.300 to get in the degradation chamber. 00:14:59.300 --> 00:15:01.240 So you'd imagine you want that to be lined up 00:15:01.240 --> 00:15:05.470 quite well in order for it to be efficient there. 00:15:05.470 --> 00:15:06.400 OK. 00:15:06.400 --> 00:15:10.270 So what are these triple-A-plus ATPases? 00:15:13.330 --> 00:15:18.620 This is a very important group of ATPases. 00:15:18.620 --> 00:15:22.180 So what the name means, ATPases associated 00:15:22.180 --> 00:15:24.670 with various cellular activities. 00:15:24.670 --> 00:15:27.250 And they're super-duper, given this. 00:15:27.250 --> 00:15:29.320 Hopefully everyone gets a triple-A-plus 00:15:29.320 --> 00:15:31.790 on the exam tonight. 00:15:31.790 --> 00:15:36.550 The superfamily is involved in many cellular functions, and-- 00:15:36.550 --> 00:15:37.310 take a look. 00:15:37.310 --> 00:15:39.150 So, many diverse functions. 00:15:39.150 --> 00:15:42.010 Cell membrane fusion, trafficking 00:15:42.010 --> 00:15:46.090 of vesicles, cytoskeleton regulation, transport, 00:15:46.090 --> 00:15:48.790 organelle biogenesis, DNA replication, 00:15:48.790 --> 00:15:50.670 transcription regulation. 00:15:50.670 --> 00:15:54.230 And what we're really interested in here is protein degradation. 00:15:54.230 --> 00:15:58.780 So they come up in a variety of processes. 00:15:58.780 --> 00:16:02.470 And although these processes are very different, 00:16:02.470 --> 00:16:04.810 all of these triple-A-plus ATPases 00:16:04.810 --> 00:16:08.530 share a common protein architecture. 00:16:08.530 --> 00:16:13.260 And I'll just point out that there's an ATP binding module. 00:16:13.260 --> 00:16:16.360 And some details are given here in terms of the motifs. 00:16:16.360 --> 00:16:19.060 And then really what we'll focus on, 00:16:19.060 --> 00:16:20.800 in terms of aspects of this course, 00:16:20.800 --> 00:16:26.230 is that they form oligomers that are ring- or cylinder-shaped. 00:16:26.230 --> 00:16:30.130 And they're all hexamers here. 00:16:30.130 --> 00:16:35.410 And so, of importance to ClpXP, these ATPases 00:16:35.410 --> 00:16:38.980 have the ability to remodel conformation of macromolecules. 00:16:38.980 --> 00:16:40.682 And so here we're focused on unfolding. 00:16:40.682 --> 00:16:41.182 Yeah. 00:16:41.182 --> 00:16:42.849 AUDIENCE: [INAUDIBLE] question, but what 00:16:42.849 --> 00:16:44.790 are ATPases that aren't associated 00:16:44.790 --> 00:16:46.090 with cellular activities? 00:16:48.650 --> 00:16:50.480 PROFESSOR: Well-- [LAUGHS] [INAUDIBLE] 00:16:50.480 --> 00:16:55.270 AUDIENCE: Is this definition based on the architecture? 00:16:55.270 --> 00:16:56.490 PROFESSOR: Strictly, yeah. 00:16:56.490 --> 00:16:57.198 I mean, there's-- 00:16:57.198 --> 00:17:00.060 GUEST SPEAKER: If you look at tRNA synthetases, 00:17:00.060 --> 00:17:01.550 they have ATPase activity. 00:17:01.550 --> 00:17:03.992 Hundreds of proteins have ATPase activities. 00:17:03.992 --> 00:17:04.700 PROFESSOR: Right. 00:17:04.700 --> 00:17:05.260 So-- 00:17:05.260 --> 00:17:07.430 GUEST SPEAKER: They hydrolyze ATP to ADP and Pi. 00:17:07.430 --> 00:17:08.680 AUDIENCE: Right, right, right. 00:17:08.680 --> 00:17:10.770 But that's a cellular activity, right? 00:17:10.770 --> 00:17:17.420 AUDIENCE: So, like, what aren't AAA-plus ATPases? 00:17:17.420 --> 00:17:20.250 PROFESSOR: Well, aminoacyl-tRNA synthetases 00:17:20.250 --> 00:17:23.300 are not triple-A-plus ATPase. 00:17:23.300 --> 00:17:25.670 What we'll see in terms of the non-ribosomal peptide 00:17:25.670 --> 00:17:29.220 synthetases, they're not these triple-A-plus ATPases. 00:17:29.220 --> 00:17:29.720 Right? 00:17:29.720 --> 00:17:31.580 So these-- I mean, yes, OK. 00:17:31.580 --> 00:17:33.860 All ATPases, the enzymes in a cell, 00:17:33.860 --> 00:17:37.310 it's going to have some role in a cellular activity, right? 00:17:37.310 --> 00:17:39.830 So maybe this name isn't very helpful. 00:17:39.830 --> 00:17:41.810 But what's common about all of these 00:17:41.810 --> 00:17:44.300 is that they share this common structural motif. 00:17:44.300 --> 00:17:46.057 They form these hexamers. 00:17:46.057 --> 00:17:48.140 But within that, there's quite a bit of diversity, 00:17:48.140 --> 00:17:50.140 because they have all these different functions. 00:17:50.140 --> 00:17:53.480 So we can just see that here, to some degree. 00:17:53.480 --> 00:17:54.760 So-- oops. 00:17:54.760 --> 00:17:57.680 And there's a typo, which I'll fix before posting. 00:17:57.680 --> 00:18:02.030 But if we take a look just at two examples here of different 00:18:02.030 --> 00:18:05.300 hexameric rings-- so these are two different triple-A-plus 00:18:05.300 --> 00:18:07.370 ATPases-- 00:18:07.370 --> 00:18:08.660 what do we see? 00:18:08.660 --> 00:18:12.170 So in common, they're both hexamers. 00:18:12.170 --> 00:18:17.750 In common, they both have an axial pore here. 00:18:17.750 --> 00:18:21.440 But we see different elements of secondary structure. 00:18:21.440 --> 00:18:22.973 And granted, these are both depicted 00:18:22.973 --> 00:18:24.140 in a bit of a different way. 00:18:24.140 --> 00:18:25.548 But if we look here-- 00:18:25.548 --> 00:18:26.090 I mean, look. 00:18:26.090 --> 00:18:29.900 We have these alpha helical regions around the exterior 00:18:29.900 --> 00:18:32.750 that we don't see here. 00:18:32.750 --> 00:18:34.100 And in this view-- 00:18:34.100 --> 00:18:36.860 I show this particular one because it's depicted 00:18:36.860 --> 00:18:38.820 where the ATP is binding. 00:18:38.820 --> 00:18:41.660 So you can see the ATP binding to each subunit here. 00:18:41.660 --> 00:18:46.400 So, as shown, six ATPs bound. 00:18:46.400 --> 00:18:49.290 So the structural diversity is quite tremendous. 00:18:49.290 --> 00:18:51.150 And here's just another example. 00:18:51.150 --> 00:18:55.310 So these are three different triple-A-plus ATPases 00:18:55.310 --> 00:18:56.390 of the Clp system. 00:18:56.390 --> 00:19:01.080 So we're going to focus on ClpX, but it's not the only one. 00:19:01.080 --> 00:19:04.190 And so what we're looking at here is ClpX. 00:19:04.190 --> 00:19:09.140 We have another family member, ClpA, and here, ClpB. 00:19:09.140 --> 00:19:12.080 And so what we see is, subunit to subunit, 00:19:12.080 --> 00:19:16.110 whether it's X, A, or B, there's quite a bit of difference, 00:19:16.110 --> 00:19:16.610 right? 00:19:16.610 --> 00:19:20.390 So ClpX is the most simple, in terms of the architecture here, 00:19:20.390 --> 00:19:22.450 for that. 00:19:22.450 --> 00:19:24.680 And so one thing people think about is, 00:19:24.680 --> 00:19:26.300 in terms of the different activities 00:19:26.300 --> 00:19:29.150 that have been associated with these different family members, 00:19:29.150 --> 00:19:31.510 how is it that these different structural features play 00:19:31.510 --> 00:19:32.366 a role? 00:19:32.366 --> 00:19:33.340 OK. 00:19:33.340 --> 00:19:35.120 Here. 00:19:35.120 --> 00:19:45.170 So, coming back to ClpX and the depiction we saw before, 00:19:45.170 --> 00:19:48.140 ClpX is an unfoldase. 00:19:48.140 --> 00:19:50.930 And what's really a key point here 00:19:50.930 --> 00:19:55.640 is that ATP hydrolysis by ClpX is 00:19:55.640 --> 00:19:58.610 going to power conformational changes that 00:19:58.610 --> 00:20:03.080 allow for mechanical unfolding of this protein that's 00:20:03.080 --> 00:20:07.130 condemned for degradation by ClpP. 00:20:07.130 --> 00:20:10.100 And that's what's going to also allow for translocation 00:20:10.100 --> 00:20:12.740 of the resulting unfolded protein 00:20:12.740 --> 00:20:14.780 into the degradation chamber. 00:20:14.780 --> 00:20:17.690 So the action of ClpX is allowing that protein 00:20:17.690 --> 00:20:20.120 to fit through this axial pore and be 00:20:20.120 --> 00:20:21.590 threaded into the chamber. 00:20:24.470 --> 00:20:27.110 So with that, what are the questions 00:20:27.110 --> 00:20:30.980 we need to address in thinking about how this macromolecular 00:20:30.980 --> 00:20:33.200 machine works? 00:20:33.200 --> 00:20:36.290 One, how are substrates recognized? 00:20:36.290 --> 00:20:38.510 So there's some certain group of proteins 00:20:38.510 --> 00:20:41.570 that are going to be degraded by this machinery. 00:20:41.570 --> 00:20:43.580 What is the mechanism? 00:20:43.580 --> 00:20:47.210 How is it that ATP-dependent conformational changes 00:20:47.210 --> 00:20:51.410 of ClpXP drive unfolding and translocation? 00:20:51.410 --> 00:20:53.480 And what is the substrate selectivity? 00:20:53.480 --> 00:20:56.940 So that's where we're going to move forward with. 00:20:56.940 --> 00:20:59.480 And so the first question we need to ask 00:20:59.480 --> 00:21:03.450 is, how are the substrates recognized by ClpX? 00:21:03.450 --> 00:21:04.300 OK? 00:21:04.300 --> 00:21:04.800 Here. 00:21:18.310 --> 00:21:20.420 And so, what are possibilities? 00:21:27.620 --> 00:21:28.430 OK. 00:21:28.430 --> 00:21:32.210 First, what we'll consider is a degradation tag. 00:21:39.150 --> 00:21:41.040 So when I draw these cartoons, I'm 00:21:41.040 --> 00:21:44.643 only going to show one of the two rings for ClpP. 00:21:44.643 --> 00:21:45.810 It's not that it's only one. 00:21:45.810 --> 00:21:47.730 This is just for simplicity. 00:21:47.730 --> 00:22:02.970 But imagine that here we have X, here we have P. 00:22:02.970 --> 00:22:05.610 And we have some condemned protein, which 00:22:05.610 --> 00:22:06.735 I'll just draw as a circle. 00:22:10.450 --> 00:22:12.820 So the cell no longer wants this protein. 00:22:12.820 --> 00:22:13.870 It needs to go away. 00:22:16.780 --> 00:22:19.750 And we can imagine, as one possibility, 00:22:19.750 --> 00:22:25.600 is that a degradation tag can be attached to this polypeptide. 00:22:25.600 --> 00:22:28.870 And what we find is that there's a particular tag called 00:22:28.870 --> 00:22:36.100 ssrA tag that is used to tag proteins for degradation 00:22:36.100 --> 00:22:38.170 by ClpXP. 00:22:38.170 --> 00:22:40.510 So we can think of this tag as a zip code. 00:22:43.090 --> 00:22:47.650 If a polypeptide gets modified such that this tag is appended, 00:22:47.650 --> 00:22:51.790 it's going to end up going to this degradation machine such 00:22:51.790 --> 00:22:53.680 that it gets degraded. 00:22:53.680 --> 00:22:55.675 The tag is 11 amino acids. 00:23:00.870 --> 00:23:02.700 It's attached to the C-terminus. 00:23:11.360 --> 00:23:11.880 OK. 00:23:11.880 --> 00:23:20.641 And the sequence is A, A, N, D, E, N, Y, A, L, A, 00:23:20.641 --> 00:23:29.100 A. And so what happens in this case, as shown-- 00:23:29.100 --> 00:23:35.550 we can imagine that this tag binds to the pore of ClpX 00:23:35.550 --> 00:23:36.840 directly. 00:23:36.840 --> 00:23:42.330 And the tag, when binding, enables translocation. 00:23:42.330 --> 00:23:42.900 So here-- 00:24:08.680 --> 00:24:09.180 OK. 00:24:09.180 --> 00:24:18.500 And this pore has what are termed pore loops that 00:24:18.500 --> 00:24:19.865 are involved in tag binding. 00:24:27.400 --> 00:24:33.560 And in particular, there's a region, GYVG-- 00:24:33.560 --> 00:24:36.280 so, a four-amino-acid sequence-- 00:24:36.280 --> 00:24:44.090 that is thought to grip and drag the substrate. 00:24:47.866 --> 00:24:48.790 OK? 00:24:48.790 --> 00:24:49.290 Here. 00:24:49.290 --> 00:24:51.480 And of course it's not gripping like we would, 00:24:51.480 --> 00:24:53.970 but there's some interaction there happening 00:24:53.970 --> 00:24:55.370 that allows that to occur. 00:24:55.370 --> 00:24:58.800 So you'll see there's a lot of mechanical-type cartoons 00:24:58.800 --> 00:25:02.860 and language used in describing these machines. 00:25:02.860 --> 00:25:03.360 OK. 00:25:03.360 --> 00:25:05.340 So what is another possibility? 00:25:21.420 --> 00:25:25.590 So another possibility for how an ATPase could interact 00:25:25.590 --> 00:25:39.180 with a degradation chamber is that the protein substrate 00:25:39.180 --> 00:25:49.810 binds to an extra domain attached to the ATPase. 00:25:59.590 --> 00:26:00.090 OK. 00:26:00.090 --> 00:26:03.480 And I point out, this possibility is not for ClpX, 00:26:03.480 --> 00:26:05.850 but it's one to be aware of, because it can occur. 00:26:12.680 --> 00:26:16.500 We saw some of those other ATPase are quite complicated. 00:26:16.500 --> 00:26:23.870 So in this case, imagine we have our ATPase, 00:26:23.870 --> 00:26:27.830 we have the degradation chamber. 00:26:27.830 --> 00:26:41.310 And this ATPase has some extra domain 00:26:41.310 --> 00:26:52.800 that effectively can bind the condemned protein 00:26:52.800 --> 00:26:56.490 and help deliver it to the pore here. 00:26:59.480 --> 00:27:02.320 And just as a third possibility, and something 00:27:02.320 --> 00:27:04.360 that we'll see moving forward, is 00:27:04.360 --> 00:27:07.750 that there is involvement of an adaptor protein. 00:27:07.750 --> 00:27:11.650 So in addition to the ATPase and the degradation chamber, 00:27:11.650 --> 00:27:15.290 there's an adaptor protein that comes into play. 00:27:15.290 --> 00:27:16.615 So in this case, the protein-- 00:27:29.942 --> 00:27:30.825 OK, adaptor-- 00:27:37.420 --> 00:27:37.920 OK. 00:27:37.920 --> 00:27:40.795 And this protein helps direct it to the pore-- 00:27:48.300 --> 00:27:56.905 so, the condemned protein to the ATPase. 00:27:59.300 --> 00:27:59.800 OK. 00:27:59.800 --> 00:28:06.093 So for instance, here we have the ATPase. 00:28:09.290 --> 00:28:13.460 Here we have the degradation chamber. 00:28:13.460 --> 00:28:26.370 And maybe there's some additional protein 00:28:26.370 --> 00:28:33.440 that facilitates getting the condemned 00:28:33.440 --> 00:28:35.400 protein to the ATPase. 00:28:38.060 --> 00:28:40.220 And so something to keep in mind, 00:28:40.220 --> 00:28:47.510 and what we'll see with ClpXP, is that one and three are not 00:28:47.510 --> 00:28:48.710 mutually exclusive. 00:28:56.920 --> 00:28:59.110 And there's an adaptor protein named 00:28:59.110 --> 00:29:09.310 SspB that can help deliver ssrA-tagged polypeptides 00:29:09.310 --> 00:29:14.410 or proteins to the degradation chamber here. 00:29:21.900 --> 00:29:28.550 So we're going to think about these ssrA tags quite a bit. 00:29:28.550 --> 00:29:31.040 And something else to be aware of 00:29:31.040 --> 00:29:33.320 is just that these ssrA tags are not 00:29:33.320 --> 00:29:37.083 the only ways of tagging proteins for degradation. 00:29:37.083 --> 00:29:39.500 We're not going to talk about it in detail in this course, 00:29:39.500 --> 00:29:41.420 but you should be aware of something 00:29:41.420 --> 00:29:44.120 called the N-end rule. 00:29:44.120 --> 00:29:45.290 And this is really cool. 00:29:45.290 --> 00:29:51.140 So this rule basically states that a half-life of a protein 00:29:51.140 --> 00:29:54.290 is determined by its N-terminal residue here. 00:29:54.290 --> 00:29:57.110 And this can be called an N-degron. 00:29:57.110 --> 00:30:01.640 And these N-degrons are recognized by proteins 00:30:01.640 --> 00:30:03.860 such as ClpS and E. coli. 00:30:03.860 --> 00:30:08.120 And as a result, these proteins can get delivered 00:30:08.120 --> 00:30:09.630 to degradation machines. 00:30:09.630 --> 00:30:13.070 So for instance, in addition to ClpXP, 00:30:13.070 --> 00:30:17.510 there's an ATPase, ClpA, that can associate with ClpP 00:30:17.510 --> 00:30:20.840 and be involved in degradation of polypeptides 00:30:20.840 --> 00:30:23.060 via this N-end rule. 00:30:23.060 --> 00:30:24.590 And in terms of the rule, depending 00:30:24.590 --> 00:30:27.470 on the identity of this N-terminal amino acid, 00:30:27.470 --> 00:30:30.530 it may be stabilizing or destabilizing, in terms 00:30:30.530 --> 00:30:32.200 of protein lifetime. 00:30:32.200 --> 00:30:33.950 If you're curious to know more about that, 00:30:33.950 --> 00:30:38.210 we can refer you to some literature. 00:30:38.210 --> 00:30:43.820 So here we have a cartoon looking 00:30:43.820 --> 00:30:48.470 at a native protein substrate that needs to be degraded. 00:30:48.470 --> 00:30:50.570 It's been modified with a tag. 00:30:50.570 --> 00:30:52.130 We have ClpX here. 00:30:52.130 --> 00:30:54.160 We have ClpP. 00:30:54.160 --> 00:30:57.380 Here's the tag. 00:30:57.380 --> 00:30:59.890 And in addition, we can have this adaptor protein 00:30:59.890 --> 00:31:04.340 SspB and the adaptor ClpS. 00:31:04.340 --> 00:31:07.760 So let's think about this tag for a minute. 00:31:07.760 --> 00:31:09.320 And we need to think about this tag 00:31:09.320 --> 00:31:12.920 from the standpoint, one, of in vitro experiments, 00:31:12.920 --> 00:31:15.830 because we're going to begin to look at some experiments that 00:31:15.830 --> 00:31:19.220 were done to understand how this machine works. 00:31:19.220 --> 00:31:21.770 And we also need to think about this tag 00:31:21.770 --> 00:31:25.470 from the standpoint of the cell. 00:31:25.470 --> 00:31:29.900 So if we think about an in vitro experiment where 00:31:29.900 --> 00:31:34.670 we want to study how ClpXP degrades some protein 00:31:34.670 --> 00:31:38.810 substrate, we can use this ssrA tag. 00:31:38.810 --> 00:31:43.520 And it's quite easy to attach 11 amino acids to some protein 00:31:43.520 --> 00:31:46.670 or polypeptide at the C-terminus. 00:31:46.670 --> 00:31:48.470 We can do that by protein expression, 00:31:48.470 --> 00:31:52.280 we can do that by chemical synthesis here. 00:31:52.280 --> 00:31:55.220 And so we're going to look at a number of experiments 00:31:55.220 --> 00:31:58.950 where this ssrA tag has been appended to certain model 00:31:58.950 --> 00:32:02.250 substrates, moving forward. 00:32:02.250 --> 00:32:04.980 So what about in the cell? 00:32:04.980 --> 00:32:08.600 So when is this ssrA tag attached to a protein? 00:32:13.250 --> 00:32:19.520 So are all proteins that need to be degraded destined to ClpXP? 00:32:19.520 --> 00:32:23.410 Just intuitively, what do you think? 00:32:23.410 --> 00:32:24.870 I see shaking heads, no. 00:32:24.870 --> 00:32:25.570 Right? 00:32:25.570 --> 00:32:29.040 There's many, many proteases around. 00:32:29.040 --> 00:32:35.952 So what proteins are destined for degradation by ClpXP? 00:32:35.952 --> 00:32:37.410 That's what we're going to look at, 00:32:37.410 --> 00:32:41.310 and how this tag is appended. 00:32:41.310 --> 00:32:50.610 And so effectively, this ssrA tag, say, in E. coli, is used-- 00:32:50.610 --> 00:32:54.960 one, because protein degradation needs to be tightly regulated. 00:32:54.960 --> 00:32:59.950 But two, it's used for dealing with proteins that 00:32:59.950 --> 00:33:02.530 exhibited stalled translation. 00:33:02.530 --> 00:33:06.290 So this discussion is going to bring us back to the ribosome 00:33:06.290 --> 00:33:06.790 here. 00:33:09.880 --> 00:33:12.220 So we want to ask what proteins in the cell 00:33:12.220 --> 00:33:15.340 are tagged with ssrA. 00:33:15.340 --> 00:33:19.490 How is the tag attached to the [INAUDIBLE] protein as well 00:33:19.490 --> 00:33:21.930 here? 00:33:21.930 --> 00:33:24.360 This is just a cartoon showing an adaptor protein 00:33:24.360 --> 00:33:31.080 helping direct this tag to the substrate here. 00:33:31.080 --> 00:33:36.090 So we're going to just move forward to this slide. 00:33:36.090 --> 00:33:39.960 This tag is specifically added to proteins 00:33:39.960 --> 00:33:43.710 that are experiencing stalled translation. 00:33:43.710 --> 00:33:46.620 So it's estimated that on the order 00:33:46.620 --> 00:33:51.960 of 0.5% of E. coli translations result in ssrA tagging. 00:33:51.960 --> 00:33:56.220 And so this is thought to be one largely of quality control. 00:33:56.220 --> 00:33:59.130 So you can imagine, if the ribosome stalled, 00:33:59.130 --> 00:34:02.370 there could be buildup of peptide products 00:34:02.370 --> 00:34:04.530 that aren't wanted. 00:34:04.530 --> 00:34:06.930 And the translation machinery could get blocked, 00:34:06.930 --> 00:34:09.679 and we don't really want that to happen here. 00:34:12.199 --> 00:34:16.150 So here's our friend, the ribosome. 00:34:16.150 --> 00:34:19.960 And here's looking at the 50S ribosomal subunit. 00:34:19.960 --> 00:34:23.050 And we have a polypeptide emerging from the exit tunnel. 00:34:23.050 --> 00:34:25.480 So these should all be familiar at this stage. 00:34:25.480 --> 00:34:27.520 And so what happens when this ribosome is 00:34:27.520 --> 00:34:29.949 trying to synthesize a polypeptide 00:34:29.949 --> 00:34:32.020 and it just gets stuck? 00:34:32.020 --> 00:34:37.989 So this ssrA tag is attached to the C-terminus of proteins. 00:34:37.989 --> 00:34:40.900 And as we're going to see, this occurs cotranslationally. 00:34:40.900 --> 00:34:47.120 And it's very, very interesting machinery. 00:34:47.120 --> 00:34:51.639 So what we see here is that there's 00:34:51.639 --> 00:34:54.400 a new player we haven't yet seen. 00:34:54.400 --> 00:34:59.200 And this is called ssrA, or tmRNA, 00:34:59.200 --> 00:35:02.470 for transfer messenger RNA. 00:35:02.470 --> 00:35:06.130 And it's involved in attachment of this ssrA tag 00:35:06.130 --> 00:35:09.960 to polypeptides that are having stalled 00:35:09.960 --> 00:35:12.190 biosynthesis on the ribosome. 00:35:12.190 --> 00:35:18.130 And so this player acts as both a tRNA and an mRNA. 00:35:18.130 --> 00:35:20.840 And we can take a look at the structure shown here. 00:35:20.840 --> 00:35:25.180 So here we have tRNA in the cloverleaf depiction, just 00:35:25.180 --> 00:35:27.250 an Ala-tRNA Ala. 00:35:27.250 --> 00:35:31.000 And if we take a look here, what do we see? 00:35:31.000 --> 00:35:34.870 At this end we have a region of the tmRNA 00:35:34.870 --> 00:35:37.350 that looks like a tRNA. 00:35:37.350 --> 00:35:37.850 Right? 00:35:37.850 --> 00:35:42.530 Quite similar here to the [INAUDIBLE] prime end. 00:35:42.530 --> 00:35:46.800 And then we have this additional region. 00:35:46.800 --> 00:35:50.890 And then if we look down in here, what do we see? 00:35:50.890 --> 00:35:53.040 We see a region that, with a little imagination, 00:35:53.040 --> 00:35:55.920 we can think looks like mRNA. 00:35:55.920 --> 00:36:00.570 And if we take a look at the various codons, what we see 00:36:00.570 --> 00:36:08.490 is that the ssrA tag is encoded there, along with a stop codon. 00:36:08.490 --> 00:36:11.490 So effectively we have a tRNA look-alike. 00:36:11.490 --> 00:36:15.825 We have an mRNA look-alike that is encoding this ssrA tag. 00:36:19.830 --> 00:36:22.850 So what happens? 00:36:22.850 --> 00:36:28.710 There's a partner protein called smpB just to be aware of. 00:36:28.710 --> 00:36:33.690 And in complex with smpB, it's actually 00:36:33.690 --> 00:36:42.840 EF-Tu that delivers this tmRNA to the ribosome here. 00:36:42.840 --> 00:36:46.320 So this is pretty interesting, just from the standpoint 00:36:46.320 --> 00:36:48.540 of what we know about EF-Tu. 00:36:48.540 --> 00:36:53.310 We don't have a typical anticodon here. 00:36:53.310 --> 00:36:54.622 So how does that happen? 00:36:54.622 --> 00:36:56.080 We're not going to go into details, 00:36:56.080 --> 00:36:57.780 but it's something-- you know, curiosity 00:36:57.780 --> 00:37:01.090 should beg those questions. 00:37:01.090 --> 00:37:03.600 So what happens? 00:37:03.600 --> 00:37:07.590 We can look at this cartoon overview here. 00:37:07.590 --> 00:37:09.750 And so the color coding within this 00:37:09.750 --> 00:37:12.930 is helpful, in terms of keeping track of pieces. 00:37:12.930 --> 00:37:15.600 But here we start with our stalled ribosome. 00:37:15.600 --> 00:37:17.550 So the mRNA is bound. 00:37:17.550 --> 00:37:21.310 We see there's a peptidyl tRNA in the P-site. 00:37:21.310 --> 00:37:24.780 You know the polypeptide has a number of amino acids, 00:37:24.780 --> 00:37:26.640 and the A-site is empty. 00:37:26.640 --> 00:37:31.620 And for some reason, no new aminoacyl tRNA is coming in. 00:37:31.620 --> 00:37:34.440 So the ribosome stalls. 00:37:34.440 --> 00:37:40.140 And as a result, this ssrA, or tmRNA, 00:37:40.140 --> 00:37:43.080 is recruited to this stalled ribosome. 00:37:43.080 --> 00:37:45.840 And so here we see the tRNA end in yellow, 00:37:45.840 --> 00:37:46.935 with the alanine attached. 00:37:46.935 --> 00:37:49.980 And here we have that region that's 00:37:49.980 --> 00:37:52.650 mRNA-like encoding the tag. 00:37:52.650 --> 00:37:55.920 So this biomolecule gets recruited. 00:37:55.920 --> 00:37:57.210 And what do we see? 00:37:57.210 --> 00:37:59.610 It enters the A-site. 00:37:59.610 --> 00:38:03.690 So here we see the tRNA end in A-site, 00:38:03.690 --> 00:38:06.900 and we have the rest of the molecule here. 00:38:06.900 --> 00:38:08.340 Then what? 00:38:08.340 --> 00:38:11.140 There's formation of a new peptide bond, 00:38:11.140 --> 00:38:12.780 so we have peptidyl transfer. 00:38:12.780 --> 00:38:14.290 We see that alanine is here. 00:38:14.290 --> 00:38:14.790 Look. 00:38:14.790 --> 00:38:16.665 That looks quite a bit like the hybrid states 00:38:16.665 --> 00:38:21.000 we talked about, where we're seeing these ends 00:38:21.000 --> 00:38:25.080 shift into the E-site, not shown, and the P-site here. 00:38:25.080 --> 00:38:26.340 And then what happens? 00:38:26.340 --> 00:38:29.790 There's translocation and there's message switching. 00:38:29.790 --> 00:38:33.690 So the original mRNA gets kicked out. 00:38:33.690 --> 00:38:35.280 And what do we see? 00:38:35.280 --> 00:38:42.970 Now that mRNA-like region of the tRNA is in A-site here. 00:38:42.970 --> 00:38:46.480 Then what happens after replacement of the mRNA? 00:38:46.480 --> 00:38:48.670 Translation can occur, which results 00:38:48.670 --> 00:38:51.760 in synthesis of the ssrA tag. 00:38:51.760 --> 00:38:54.910 So that's how this tag is attached to the C-terminus 00:38:54.910 --> 00:38:57.300 of the polypeptide. 00:38:57.300 --> 00:39:01.240 And elongation occurs until that stop codon in the tmRNA 00:39:01.240 --> 00:39:03.250 enters the A-site. 00:39:03.250 --> 00:39:06.100 And then peptide release occurs here. 00:39:06.100 --> 00:39:09.880 So the result is a protein that has the ssrA 00:39:09.880 --> 00:39:12.340 tag attached to its C-terminus. 00:39:12.340 --> 00:39:18.070 And that protein will be directed to ClpXP. 00:39:18.070 --> 00:39:20.120 So, pretty cool. 00:39:20.120 --> 00:39:20.620 Yeah. 00:39:20.620 --> 00:39:22.210 I think so. 00:39:22.210 --> 00:39:22.840 There. 00:39:22.840 --> 00:39:28.300 We don't ever leave the ribosome too much within these units. 00:39:28.300 --> 00:39:32.590 Just to point out, this tag is universal in bacteria. 00:39:32.590 --> 00:39:36.370 So here's just a table of phylogenetic distribution. 00:39:36.370 --> 00:39:40.250 You're not responsible for these details. 00:39:40.250 --> 00:39:41.110 Yeah. 00:39:41.110 --> 00:39:43.180 AUDIENCE: About the last slide-- 00:39:43.180 --> 00:39:47.630 so is the tag attached after it's stalled? 00:39:47.630 --> 00:39:49.780 Like, is the original protein completed? 00:39:49.780 --> 00:39:55.400 Or just the original mRNA removed and detached the tag? 00:39:55.400 --> 00:39:56.313 Or it's both? 00:39:56.313 --> 00:39:56.980 PROFESSOR: Yeah. 00:39:56.980 --> 00:40:00.380 So what does the cartoon suggest? 00:40:00.380 --> 00:40:02.843 AUDIENCE: It feels like it's already on the C-end here. 00:40:06.550 --> 00:40:11.620 PROFESSOR: Well, the ribosome can stall at various points. 00:40:11.620 --> 00:40:14.830 So imagine you have a 100-amino-acid polypeptide that 00:40:14.830 --> 00:40:16.990 needs to be synthesized. 00:40:16.990 --> 00:40:22.600 The ribosome could stall after amino acid 20 or 40 or 60. 00:40:22.600 --> 00:40:24.280 It's not that the whole polypeptide 00:40:24.280 --> 00:40:28.520 has been synthesized and then this gets put on. 00:40:28.520 --> 00:40:33.310 It may be some fragment there for that. 00:40:33.310 --> 00:40:36.320 So, yes. 00:40:39.800 --> 00:40:43.160 So in terms of this adaptor protein, 00:40:43.160 --> 00:40:48.080 I just want to make a note in terms of the adaptor. 00:40:48.080 --> 00:40:52.220 So these adaptor proteins can help 00:40:52.220 --> 00:40:55.700 with regulating the substrate specificity 00:40:55.700 --> 00:40:58.970 of triple-A-plus ATPases. 00:40:58.970 --> 00:41:03.260 And effectively, depending on the system and depending 00:41:03.260 --> 00:41:08.280 on the adaptor, it may enhance or it may inhibit degradation 00:41:08.280 --> 00:41:08.780 here. 00:41:08.780 --> 00:41:10.220 So it's a case-by-case basis. 00:41:12.950 --> 00:41:17.570 This SspB shown in the cartoon here 00:41:17.570 --> 00:41:21.800 is a dimeric adaptor for XP, and it promotes degradation 00:41:21.800 --> 00:41:23.690 of certain substrates. 00:41:23.690 --> 00:41:28.730 And effectively, it enhances recognition of this tag 00:41:28.730 --> 00:41:33.380 by the machinery such that the degradation rates are enhanced. 00:41:33.380 --> 00:41:35.940 So it's not that it's required. 00:41:35.940 --> 00:41:38.210 It's just helpful, and accelerates the process. 00:41:58.260 --> 00:42:03.780 So just an interesting observation regarding SspB-- 00:42:03.780 --> 00:42:08.400 it can be co-purified with ribosomes here. 00:42:08.400 --> 00:42:10.920 And in terms of its structure, it 00:42:10.920 --> 00:42:15.570 has some resemblance to known RNA-binding proteins. 00:42:15.570 --> 00:42:18.960 And this resemblance has begged a question, 00:42:18.960 --> 00:42:24.450 does SspB itself help with linking protein synthesis 00:42:24.450 --> 00:42:26.850 and protein degradation? 00:42:26.850 --> 00:42:31.770 So is it possible that SspB could 00:42:31.770 --> 00:42:37.050 help promote binding of ClpX to polypeptides 00:42:37.050 --> 00:42:38.760 before full release to the ribosome? 00:42:38.760 --> 00:42:41.100 That's something people have wondered about. 00:42:41.100 --> 00:42:44.430 And initially this protein was classified 00:42:44.430 --> 00:42:47.040 as a stringent starvation protein. 00:42:47.040 --> 00:42:49.270 That's where the name comes from. 00:42:49.270 --> 00:42:53.650 So if we just take a quick look at its structure here, 00:42:53.650 --> 00:42:55.050 what do we see? 00:42:55.050 --> 00:42:57.390 So here is SspB. 00:42:57.390 --> 00:43:03.570 And then here we have structures from ribosomal proteins. 00:43:03.570 --> 00:43:08.970 And so in SspB, ClpX binds on this side. 00:43:08.970 --> 00:43:15.300 And effectively, here we look at the ribosomal proteins 00:43:15.300 --> 00:43:16.200 that bind RNA. 00:43:16.200 --> 00:43:18.900 And they have these RNA-binding sites there. 00:43:18.900 --> 00:43:21.540 So there's some similarities in terms of the alpha helix, 00:43:21.540 --> 00:43:23.330 in terms of the beta sheets here. 00:43:26.790 --> 00:43:29.100 And also, I'll just note, in terms 00:43:29.100 --> 00:43:38.260 of SspB and the ssrA tag-- 00:43:38.260 --> 00:43:41.130 so if we take this tag-- 00:43:51.230 --> 00:43:55.910 So this is our ssrA tag here. 00:43:55.910 --> 00:44:03.350 What's found is that ClpX recognition is on this end 00:44:03.350 --> 00:44:06.980 and SspB binding is on this end here. 00:44:19.190 --> 00:44:21.050 So in different points. 00:44:21.050 --> 00:44:31.790 So what this indicates here is that SspB and ClpX 00:44:31.790 --> 00:44:33.410 can bind simultaneously. 00:44:41.770 --> 00:44:42.850 OK. 00:44:42.850 --> 00:44:45.850 But this is small, so we can expect that there's 00:44:45.850 --> 00:44:49.520 some clash here for that. 00:44:55.480 --> 00:44:58.000 So where we're going to close is just 00:44:58.000 --> 00:45:02.290 looking at an overview as to how this machine works, 00:45:02.290 --> 00:45:05.710 and the model that then, starting on Friday, 00:45:05.710 --> 00:45:07.750 we'll look at experiments that were designed 00:45:07.750 --> 00:45:12.370 and performed to inform this model. 00:45:12.370 --> 00:45:16.150 So if we look at this in one type of cartoon, 00:45:16.150 --> 00:45:18.740 what are the stages? 00:45:18.740 --> 00:45:22.990 So we can think of three as depicted here, 00:45:22.990 --> 00:45:25.840 where there's some sort of initial recognition. 00:45:25.840 --> 00:45:31.060 So the ssrA tag of this condemned protein 00:45:31.060 --> 00:45:34.210 binds to the axial pore of ClpX. 00:45:34.210 --> 00:45:39.490 And this process does not require ATP hydrolysis. 00:45:39.490 --> 00:45:41.860 So here we see a folded substrate. 00:45:41.860 --> 00:45:47.230 This degron is another word for one of these tags, ssrA tag. 00:45:47.230 --> 00:45:50.800 So we see there's recognition here. 00:45:50.800 --> 00:45:55.100 Then what happens, ClpX unfolds this substrate. 00:45:55.100 --> 00:45:57.910 So somehow it has to grip and pull and apply 00:45:57.910 --> 00:46:00.580 a force that unfolds the polypeptide, 00:46:00.580 --> 00:46:03.370 and threads that unfolded polypeptide 00:46:03.370 --> 00:46:04.960 into the degradation chamber. 00:46:04.960 --> 00:46:08.330 So, you know, kind of this pulley system is shown here. 00:46:08.330 --> 00:46:11.480 This chopper-type thing is shown here. 00:46:11.480 --> 00:46:14.500 You can use your imagination in this unit 00:46:14.500 --> 00:46:16.630 for how to depict this machine. 00:46:16.630 --> 00:46:20.560 So we see that the polypeptide is being unfolded and threaded 00:46:20.560 --> 00:46:22.630 through ClpX into this chamber, where 00:46:22.630 --> 00:46:28.660 it gets chopped up by the serine protease active sites here. 00:46:28.660 --> 00:46:32.990 So for unfolding and translocation, ATP is needed. 00:46:32.990 --> 00:46:37.420 ClpX is hydrolyzing ATP to allow this to occur. 00:46:37.420 --> 00:46:39.970 In the degradation chamber, this degradation part 00:46:39.970 --> 00:46:41.270 is independent of ATP. 00:46:41.270 --> 00:46:41.770 Right? 00:46:41.770 --> 00:46:45.140 The serine protease doesn't need that here. 00:46:45.140 --> 00:46:47.830 So how can we kind of break this up further into a model 00:46:47.830 --> 00:46:49.600 that we can test? 00:46:49.600 --> 00:46:52.600 What I present here is the working model. 00:46:52.600 --> 00:46:56.320 And just note, the orientation is flipped here. 00:46:56.320 --> 00:47:00.910 So we have ClpX on the bottom and ClpP on top. 00:47:00.910 --> 00:47:04.270 So what happens here? 00:47:04.270 --> 00:47:08.380 We can look at this in terms of five steps. 00:47:08.380 --> 00:47:11.680 And we can begin here, with binding. 00:47:11.680 --> 00:47:16.150 So this ssrA-tagged protein needs to bind to ClpX. 00:47:16.150 --> 00:47:19.000 And that binding is associated with a dissociation 00:47:19.000 --> 00:47:23.050 constant, or Kd here. 00:47:23.050 --> 00:47:24.160 What do we see? 00:47:24.160 --> 00:47:28.930 After binding we have a second step, which is denaturation. 00:47:28.930 --> 00:47:33.550 So the polypeptide becomes unfolded. 00:47:33.550 --> 00:47:37.900 And that's defined by a rate constant for denaturation, 00:47:37.900 --> 00:47:40.220 as shown here. 00:47:40.220 --> 00:47:43.030 If we look next, we have translocation. 00:47:43.030 --> 00:47:46.840 So this polypeptide is moving through ClpX 00:47:46.840 --> 00:47:49.570 into the degradation chamber. 00:47:49.570 --> 00:47:53.230 And this is also associated with the rate constant-- 00:47:53.230 --> 00:47:55.450 so, rate constant for translocation. 00:47:55.450 --> 00:48:01.120 And both of these steps require the use of ATP. 00:48:01.120 --> 00:48:03.490 Once this polypeptide is in the chamber, 00:48:03.490 --> 00:48:07.410 we have step four, which is degradation. 00:48:07.410 --> 00:48:10.150 And again, we have k deg. 00:48:10.150 --> 00:48:11.440 This is fast. 00:48:11.440 --> 00:48:14.860 And then in this last step here, there's some release. 00:48:14.860 --> 00:48:16.900 So somehow these polypeptide fragments 00:48:16.900 --> 00:48:19.078 need to be released from the chamber. 00:48:19.078 --> 00:48:21.890 AUDIENCE: Is ClpP still a dimer at this point? 00:48:21.890 --> 00:48:22.880 PROFESSOR: Yes, yes. 00:48:22.880 --> 00:48:25.120 So often the cartoons are drawn just 00:48:25.120 --> 00:48:28.810 showing one of the heptamers. 00:48:28.810 --> 00:48:32.050 But think of it as a dimer, with these two back-to-back rings 00:48:32.050 --> 00:48:35.030 here for that. 00:48:35.030 --> 00:48:35.530 Right. 00:48:35.530 --> 00:48:38.830 So we have five steps here. 00:48:38.830 --> 00:48:41.650 Each one of these steps has a rate constant. 00:48:41.650 --> 00:48:45.220 And one question we want to ask with this is, what 00:48:45.220 --> 00:48:47.480 is the rate-determining step? 00:48:47.480 --> 00:48:50.680 And the quick answer where we'll end today, 00:48:50.680 --> 00:48:53.560 and as indicated in this overview, 00:48:53.560 --> 00:48:58.870 is that degradation is fast relative to denaturation 00:48:58.870 --> 00:49:00.990 and translocation. 00:49:00.990 --> 00:49:03.190 And there should be an intuitive aspect to that. 00:49:03.190 --> 00:49:05.530 We heard about last time how proteases 00:49:05.530 --> 00:49:08.230 give these tremendous rate accelerations. 00:49:08.230 --> 00:49:10.510 And if you have an unfolded peptide, 00:49:10.510 --> 00:49:12.370 those sites where cleavage will happen 00:49:12.370 --> 00:49:15.580 are going to be exposed there. 00:49:15.580 --> 00:49:17.860 So what we're going to ask is, is 00:49:17.860 --> 00:49:22.660 it possible to make experiments, design experience, where 00:49:22.660 --> 00:49:28.000 we can separate the denaturation process from the translocation 00:49:28.000 --> 00:49:30.970 process and analyze those-- 00:49:30.970 --> 00:49:33.910 and in the process of doing so, ask, 00:49:33.910 --> 00:49:36.440 what is the ATP utilization for each step? 00:49:36.440 --> 00:49:40.730 And what is the role for ATP in this process? 00:49:40.730 --> 00:49:44.130 And so on Friday we'll begin with discussing 00:49:44.130 --> 00:49:47.760 substrates, the design of substrates that have been used, 00:49:47.760 --> 00:49:50.340 to examine this model in more detail. 00:49:50.340 --> 00:49:51.800 Is there one question next? 00:49:51.800 --> 00:49:54.680 AUDIENCE: I was just wondering if the degradation step also 00:49:54.680 --> 00:49:58.880 removes translational modifications, or [INAUDIBLE] 00:49:58.880 --> 00:50:01.060 PROFESSOR: In the degradation step? 00:50:01.060 --> 00:50:02.460 That's going to depend. 00:50:02.460 --> 00:50:05.850 I mean, you can have different types of bonds 00:50:05.850 --> 00:50:08.650 with post-translational modifications, right? 00:50:08.650 --> 00:50:09.150 Right. 00:50:09.150 --> 00:50:10.950 So in the eukaryotic system, you have 00:50:10.950 --> 00:50:14.100 a post-translational modification 00:50:14.100 --> 00:50:17.070 to direct this condemned protein. 00:50:17.070 --> 00:50:19.650 And that machinery-- so they're ubiquitins, 00:50:19.650 --> 00:50:21.540 and you get this polyubiquitin tail. 00:50:21.540 --> 00:50:24.180 So you saw ubiquitin in recitation number one. 00:50:24.180 --> 00:50:26.400 And the eukaryotic proteasome has the ability 00:50:26.400 --> 00:50:29.250 to chop those ubiquitins ends off 00:50:29.250 --> 00:50:31.660 for recycling there, in that. 00:50:31.660 --> 00:50:34.070 So that's one example.