Structure of Outer Membrane Protein OmpG (and how we got there) Lukas Tamm University of Virginia

Transcription

1 Structure of Outer Membrane Protein OmpG (and how we got there) Lukas Tamm University of Virginia

2 MEMBRANE PROTEINS ARE ABUNDANT AND IMPORTANT BUT KNOWLEDGE DATABASE IS LAGGING FAR BEHIND THAT OF SOLUBLE PROTEINS ~ 30 % of all open reading frames are expected to be membrane proteins ~ 0.6 % of all structures in the PDB are membrane proteins (280/46,818) 133 unique MPs ~ 60 % of all current drugs target membrane proteins (channels, GPCRs, transporters etc.)

3 Outer Membrane Protein A (OmpA) of E. coli

4 Functions ascribed to OmpA: Structural protein: connects the outer membrane to periplasmic peptidoglycan Ion channel/pore: single channel conductance in vitro response to osmotic stress in vivo Receptor for various bacteriophages (K3, Ox2) Mediates bacterial conjugation

5 15 N- 1 H TROSY NMR spectrum of OmpA TM domain in DPC micelles at 750 MHz

6 Fold of the OmpA TM domain by NMR spectroscopy 19 kda 177 residues in DPC micelle of ~46 kda Arora et al. (2001) Nat Struct Biol 8, 334

7 Interacting Gate Side-Chains in OmpA Arg 138 Glu 52 See: Hong, Szabo, & Tamm (2006) Nature Chem. Biol. 11: Glu 128 Lys 82

8 How about NMR of larger membrane proteins? larger porins of bacterial outer membranes

9 Outer membrane protein G (OmpG) facultative porin in outer membrane of E. coli facilitates uptake of large oligosaccharides only up-regulated when LamB is down-regulated monomeric 280 residues (33 kda) engineerable ion channel potential use in biosensor development no crystal structure available when project started

10 Initial attempts resulted in poor TROSY spectra despite apparent refolded + Pr-K boiled good refolding 33 kda 28 kda following extraction and refolding protocols by Conlan and Bayley Jason Schmittschmitt Spring 2004

11 Further Optimization of Refolding Optimizing the folding of OmpG, using different methods to determine the refolding efficiency: Methods for determining refolding: SDS-PAGE band shift essay CD Trp Fluorescence Testing different conditions for NMR sample preparation: micelle type and concentration, ph etc 15 N-labeled proteins, TROSY spectra

12 DPC vs.!-og at various ph ph = mM DPC, 37ºC overnight OmpG: 25 µm refolding control Proteinase K digest 70mM!-OG, 37ºC overnight OmpG: 25 µm

13 Effect of ph on TROSY in!-og 10 mm Tris, ph 9 10 mm phosphate, ph 6.3 Lower ph leads to 20% increase in peak numbers

14 Summary of OmpG Refolding The effect of folding aids tested so far is insignificant OmpG concentration has some effect, in general, lower concentration is better for achieving a high level of refolding The best conditions: 10 mm Tris, ph mm!-og (~2%) 37ºC, overnight or longer

15 Planar bilayer (single channel) recordings of OmpG pa Time (s) Buffer: 1M KCl, 10mM Tris, ph 7.2, OmpG stock: 25 µm in 70 mm!-og, 10 mm Tris, ph 9 Applied voltage: 40 mv, sampling rate: 1kHz

16 OmpG is a porin pa 5000 Time (s) # of events ns in 1 M KCl ph 7.4 at 40 mv pa

17 15 N, 13 C, 2 H-labeled OmpG sample 15 N- 1 H TROSY with 13 C decoupling 15 N- 1 H TROSY without 13 C decoupling Triple labeling and sample refolding are successful

18 Long-term stability of OmpG in!-og The average linewidth increased by 3% after 15 hours at 40 ºC Samples show a faint whitish precipitate after several days at 40 ºC

19 Detergent exchange to DPC Fresh concentrated samples refolded in!og were diluted into DPC micelle solutions Allowed to sit overnight at room temperature Mixed OmpG/DPC/!-OG solution were concentrated in Amicon using a 30K NMWL membrane Diluted and concentrated one more round with DPC micelle containing buffer

20 15 N- 1 H TROSY of 15 N,D-OmpG exchanged into DPC measured at 500 MHz OmpG is still properly folded in DPC and general features are the same Peaks are broader probably due to the larger size of the OmpG/DPC compared to OmpG/!-OG complex

21 Triple-resonance TROSY experiments at 800 MHz HNCA, HN(CO)CA HN(CA)CB, HN(COCA)CB HNCO, HN(CA)CO Different versions of these experiments were tested for the best spectral quality

22 HNCA Strip Plot (71-92)

23 HN(CA)CB Strip Plot (71-92)

24 HNCO and HN(CA)CO Strip Plot (71-81)

25 OmpG: 33 kda, 280 residues 800 MHz TROSY 234 residues assigned, 9 partially assigned, 12 tentatively assigned = 255/280

26 C" and C! secondary chemical shifts Plot of three-bond averaged secondary chemical shifts versus residue numbers The strong negative!c " -!C # values identify 14 beta strands. The markedly positive!c " -!C # region indicates the existence of a short helix between strands 7 and 8. However, this helix it was not confirmed in the structure calculation.

27 133 long range NOEs define topology and strand connectivity of OmpG from 15 N- 1 H- 1 H NOESY-TROSY and 15 N- 15 N- 1 H TROSY-NOESY-TROSY experiments

28 NOE measurements and distance restraints 316 unique NOE distances 137 sequential 46 medium-range 133 long-range (more than 4 residues apart) 262 dihedral angle restraints 55 H-bonding pairs (220 distances)

29 Ensemble of 10 Lowest-Energy Structures w/o H-bonds with H-bonds Including H-bonds improves backbone r.m.s.d. from 2.54 ± 0.47 to 1.67 ± 0.29 Å (!-sheet and turn residues)

30 Comparison with X-ray crystal structures closest-to-mean NMR closed state (2IWW) open state (2IWV) open state (2F1C) (2JQY) ph 6.3 ph 5.6 ph 7.5 ph 5.5

31 Comparison of NMR and Crystal Structures r.m.s.d. accuracy of NMR vs. different crystal structures 1.65 to 1.71 Å

32 Backbone Dynamics by Heteronuclear NOEs

33 Ribbon Representation of Lowest Energy Conformer of OmpG (largest membrane protein - 33 kda - solved by NMR to date) Liang and Tamm (2007) PNAS 104:16140

34 What s next? Improve quality of NMR structure by measuring RDCs and PREs Can we learn more about gating from measurements of membrane protein dynamics? Specifically, can we explain ph-dependent gating? What is the role of the lipid matrix in all of this?

35 Gels to introduce weak alignments to measure Residual Dipolar Couplings of OmpG 7 % 5 % 4.3 % 3.6 % Original charged gel # Swelling in H 2 O # Cut # Dried # Re-Swelling in protein/micelle solution # Sample made # Reposition piston

36 Two types of acrylamide-based copolymer gels are used to obtain multiple alignments 1. Negatively charged gels (7%): 2-Acrylamido-2-methyl-1-propanesulfonic acid (50$S and 75$S) 2. Positively charged gels (6% and 7%): (3-Acrylamidopropyl)trimethylammonium chloride (25+M, 50+M, 75+M)

37 Change of Alignments isotropic 50-S, 13mm 92.8 Hz 95.4 Hz Hz (16.6Hz) 98.9 Hz (3.5Hz) 50-S, 15mm 50-S, 17mm Hz (8.5 Hz) Hz (5.4 Hz) 95.8 Hz (3 Hz) 95.2 Hz (-0.2 Hz)

38 HNCO based RDC experiments H N -N N-C C -C " A46 N80 RDC = 12.6 Hz -3.8 Hz -2.6 Hz RDC = Hz 1.2 Hz 0 Hz

39 Refinement of OmpA Structure with Residual Dipolar Couplings measured in two different alignments in two different (+, ) charged compressed gels 421 RDC couplings: D HN D NC D C C" plus: 90 NOE restraints 142 dih. angle restraints 68 H-bond restraints rmsd precision: 0.62 ± 0.16 rmsd accuracy: 1.11 ± 0.06 Cierpicki et al. (2006) JACS 128, 4389

40 Conformations of periplasmic turns quite well defined by RDCs type I or II type I type I or II

41 Parallel Site-Directed Spin-Labeling and Paramagnetic Relaxation Enhancement to Refine Solution Structure of OmpA spin-labels introduced at 11 sites: Solomon-Bloembergen equation: r = & K $ $ % R, * sp c c 2 h - 2 c )# '! (!" 1/ 6 I I para dia = R sp 2 exp(! R2 t) sp R2 + R2 Liang et al. (2006) JACS 128:4389

42 Correlation Between Experimental NMR Distances Measured by PRE and Best Fit Distances to Paramagnetic Centers in Crystal Structure with Grafted MTSSL Fitted Distance (Å) + N46R1 T88R1 T132R1 A11R1 M53R1 T95R1 L139R1 Diagonal ± 2Å limits Experimental Distance (Å)

43 Structures of OmpA Calculated With and Without PRE Restraints no PRE restraints 90 NOE restraints 142 dih. angle restraints 68 H bond restraints rmsd precision: 1.25 ± 0.29 rmsd accuracy: 1.62 ± PRE restraints 90 NOE restraints 142 dih. angle restraints 68 H bond restraints rmsd precision: 0.85 ± 0.17 rmsd accuracy: 1.09 ± 0.12

44 In Summary (NMR): Solution NMR has gained a significant role in structural biology of membrane proteins (now extended to 33 kda protein) Functions of membrane proteins may be studied by NMR-based dynamical studies Interactions with specific lipids and substrates may be studied in ways that may not be accomplished by crystallography NMR of membrane proteins still not easy: sample preparation continues to be difficult, new NMR methods continue to be developed and tailored for use with membrane proteins, especially "-helical membrane proteins (harder) Limitations of detergent micelles as solvents: excellent to get a good start, but also try bicelles, ss-nmr, and SDSL-EPR in bilayers for verfication

45 Thanks to: NMR Structure and Dynamics Binyong Liang Ashish Arora Ming-tao Pai Membrane Protein Folding Heedeok Hong Ricardo Flores, Sangho Park Dennis Rinehart Jörg Kleinschmidt Single Channel Recordings Ashish Arora, Heedeok Hong Binyong Liang, Luiz Salay Collaborators: John Bushweller, Tomek Cierpicki, Jeff Ellena, Gabor Szabo (U. of Virginia) Funding: NIGMS

46 Chapter on solution NMR of membrane proteins Recent review by Ashish Arora in Tamm and Liang NMR of Membrane Proteins in Solution Progress in NMR Spectroscopy 48, (2006) Wiley-VCH 2005