James Milner-White

1. James Milner-White Glasgow University, UK J.Milner-White@bio.gla.ac.uk Small Motifs(up to 8 residues) In a-helix, b-sheet and polyproline II helix the main chain conformations of successive residues are identical. But if they are not?

2. P2-7 small, hydrogen bonded, motifs that recur in proteins b-turns 19% ab motifs 14% nests 7% asx-, ST- features 7% Schellmann loops 7% b-bulges 5% ST- feature 5% asx = asp, asn; ST = ser, thr % is the proportion of aas in an average protein belonging to the motif. Motifs can overlap

3. Right- and left-handed forms All main chain motifs can occur in enantiomeric forms. e.g. : RH and LH a-helix. Interconvert them by multiplying f and y by –1.

4. H H H H H H H H H H O O O O O O O O O P10 Main Chain – Main Chain Hydrogen Bond Color key N-C-C-N-C-C-N-C-C-N-C-C-N-C-C-N-C-C-N-C-C-N-C-C-N-C-C-N-C-C O R -3 2 4 5 -4 3 CO binds to NH R residues ahead other = Invoke this color scheme in Rasmol by typing: color hbond type

5. P2-7 small, hydrogen bonded, motifs that recur in proteins b-turns 19% ab motifs 14% nests 7% asx-, ST- features 7% Schellmann loops 7% b-bulges 5% ST- feature 5% asx = asp, asn; ST = ser, thr % is the proportion of aas in an average protein belonging to the motif. Motifs can overlap

6. P5 The Schellmann loop occurs at the C-terminus of a third of all a-helices M-W 1988 JMB199, 503

7. P2 The Nest anion N N N Main chain NH groups of 3 successive residues bind an anionic atom or group. 8% of residues in proteins take part in nests. Watson & Milner-White 2002 JMB315, 171

8. Carbonyl oxygen atoms are anionic in character d+ d- C = O

9. A nest binding a carbonyl oxygen, with the hydrogen atoms of the main chain NH groups shown

10. P2 The Nest anion N N N Main chain NH groups of 3 successive residues bind an anionic atom or group. 8% of residues in proteins take part in nests. Watson & Milner-White 2002 JMB315, 171

11. P11 Two Nest Conformations Nests have two types of characteristic f,y angles for residues 1 and 2. The f,y angles for residue 3 do not affect nestconformation. gR, followed by gL = RL Average f,y: -94, -1 77, 21 (80%) gL, followed by gR = LR Average f,y: 77, 12 -82, -11 (20%) RL and LR are enantiomers, regarding their main chain atoms.

12. P11 Enantiomeric conformations 180 Positive f value conformations are more comfortable as glycines, Genetically encoded amino acids apart from glycine prefer negative f values. Left-handed a-helix y NestR 0 NestL Right-handed a-helix -180 -180 0 180 f

13. RL nests occur in all Schellmann loops

14. The nest in the Schellman loop at the C-terminus of an a-helix.

15. LR nests occur in … The oxyanion hole of trypsin-like serine proteases

16. Trypsin - PTI oxyanion hole of trypsin 197 195 Peptide bond to be cleaved ‘Substrate’ of PTI

17. An LR Nest occurs in Vancomycin P11 Is a complex glycopeptide antibiotic, with residues of alternating D and L configurations. It acts by binding to a C-terminal D-ala residue, an intermediate in bacterial cell wall synthesis. In the crystal structure of the vancomycin-acetate complex, acetate mimics the terminal D-ala carboxylate.

18. Vancomycin-acetate complex Vancomycin: spacefill Acetate: sticks The three blue nitrogen atoms of the LR nest bind the carboxylate oxygen atoms

19. Compound nests As well as simple nests, alternating RLR, RLRL, or LRL, etc., sequences occur. In these the NH groups all face the anion, resulting in a wider, compound, nest that tends to bind anionic groups rather than atoms.

20. Functionally Important Nests and Compound Nests Nest type Resno PDBcode protein Anion Ligand LR 195 trypsin O peptide LRLR 13 5p21 ras P GTP LRLR 48 1aqu estrogen sulfotransferase P pyridoxal phosphate RLR 12 1rcf flavodoxin P FMN RLR 88 1amo NADPH cyt p450 reductase P FMN RL 142 1rie Rieske iron-sulfur protein I Fe2S2 RLRLR 39 1a70 spinach ferredoxin I Fe2S2 RLRLR 9 2fdn Clostridium ferredoxin I Fe4S4 RLRLRRLR 57 1qla fumarate reductase I Fe2S2 RLLRLR 46 1cje adrenodoxin I Fe2S2 RLR 387 1qj2 CO dehydrogenasemolybdo-pterin RL 212 1az2 aldose reductase P NADP RLR 37 1rge ribonuclease O of guanine of rntd RLR 1bgs barnase P RNA LRLR 179 2nmt N-myristoyl transferase P myristoyl-CoA LRLR 119 1bo4 N-acetyl transferase P acetyl CoA RLR 129 1opr orotate P.R.transferase P PRPP RL 741 RNA polymerase b’chain - - RLR* 6 1qfu Haemagglutinin b-chain O D112 LR* 92 1cog gelsolin O D87…Ca+ RLRL* 22 1cdm calmodulin O D20 D22 D24…Ca+ In the anion column a single letter indicates that an anionic atom or group binds in the nest, and shows whether it is a carbonylor carboxylate oxygen atom (O), a phosphate group (P) or an iron-sulphur center (I).

21. P12 The P-loop The ATP/GTP binding P-loop has an LRLR compound nest, binding the b-phosphate of ATP or GTP. 10GAGGVGKS17 LRLR P21rassequence

22. P12 P-loop of ras b-phosphate GTP oxygens (Red) Five main chain NH groups (blue)

23. S S S S Fe Fe S S S Fe Fe S Fe S S S Fe S Iron-Sulfur Proteins The longest compound nests (e.g.: RLRLR) surround iron-sulfur centres such as: squares cubes These centres in proteins have a net negative charge when the extra cysteine sulphurs are considered, so are anions.

24. P12 Ferredoxin Fe4S4 (+S4) cube

25. Archaeal Ferredoxin with Fe3S4 Centre

26. EF hand, from calmodulin P13 Three carboxylates bind the Ca++ A compound nest binds the carboxylates.

27. EF hand from calmodulin 24 22 20 Ca++

28. P11 180 Left-handed a-helix gL y NestgR 0 NestgL Right-handed a-helix aR -180 -180 0 180 f

29. Potassium Channels Passing from the gRgL to the aRaL conformation the nest cavity becomes shallower; and the polypeptide is more linear. aRaL conformations are uncommon compared to gRgL, with one exception. Watson& MW 2002 JMB175 199 Potassium channels are tetramers; the potassium ions pass through a channel formed by the CO groups of 4 identical polypeptides from each subunit, with the aRaL conformation.

30. Selectivity filter Potassium=pink Morais-Cabral et al., 2001 Nature414, 37-42 43-48 Potassium channel

31. The selectivity filter of the potassium channel Potassium = pink

32. P13 Single chain of selectivity filter of potassium channel with potassiums in place. T V G Potassium = pink Y

33. P13 Each potassium in the channel is surrounded by eight carbonyl oxygens

34. Catgrips The nest (and K+ channel) conformations all have approximately alternating enantiomeric main chain residues. Are there other conformations of this sort? Yes. In polypeptides where alternate main chain CO groups bind Ca++ ions. We call them catgrips. They have bRbL or bLbR conformations, RL or LR. Average f,y angles are: bR –64, 132 bL 78, -160 Like nests, they can be, and often are, compound. Watson& MW 2002 JMB 175 199

35. Ca O O O O O O O | | | | | | | C C C C C C C C | O C C Ca | | O O Ca C O | Ca C | O Catgrips Catgrip type Resno PDBcode protein Four successive mainchain carbonyl oxygens bound to 2 Calciums oLoRoLo 352,361,370 1af0 MMP: Serralysin oLoRoLo 352,361,370 1kap MMP: alkaline protease Three alternating mainchain carbonyl oxygens bound to 1 calcium oR LoR Lo 27 2ran annexin oR LoR Lo 29 1bp2 phospholipase A2 oR LoL Ro 160 2usn MMP: stromelysin oR LoL Ro 177 1mmp MMP:matrilysin oR LoL Ro 177 1nfc MMP: fibroblast collagenase oR LoL Ro 156 1bzs MMP: neutrophil collagenase oR LoL Ro 195 1buv MMP: membrane-type collagenase Two alternating mainchain carbonyl oxygens bound to 1 calcium oR Lo 99,185,259 2ran annexin oR Lo 323 1fza fibrinogen oR Lo 160 1q1b MMP: gelatinase oL Ro 87 2tec thermitase oL Ro 79 1af4 subtilisin Carlsberg oL Ro 80 1mpt M-protease oL Ro 80 1scj subtilisin E oL Ro 80 1mee mesentericopeptidase

36. Alkaline protease Baumann et al 1993 EMBO J12, 3357

37. P14 Part of the calcium binding unit of alkaline protease Baumann et al 1993 EMBO J12, 3357

38. Phospholipase A2

39. P14 Exploring the conformations with alternating enantiomeric residues

41. Ramachandran-type plot for polypeptides with alternating enantiomeric residues Structures are rings or linear. The contours give the number of residues per ring First done for cyclic peptides by DeSantis, Morosetti & Rizzo, (1974) Macromolecules 7, 52 .

42. Why should polypeptides with alternating enantiomeric conformations occur? The situation is comparable to that studied by Ramachandran and others where successive residues (in a-helix b-sheet, etc.) have identical conformations. They do not have to have identical conformations but, for geometrical reasons, they do. It is much the same with alternating enantiomeric polypeptide conformations.

43. We have made a b-version of a database of Small Hydrogen-Bonded Motifs where you can find the motifs your protein has. http://doolittle.ibls.gla.ac.uk:9006/suraj/servlet/ProteinMotifDB

44. To Do: (I’ll be around this evening and tomorrow to assist or admire.) Open Rasmol; load your favorite protein. (Best if it has < 250 aa residues so you may want to restrict it, save the new version write pdb new.pdb and reload new.pdb.) Display in BACKBONE mode, add mainchain-mainchain H-bonds via: hbonds 30 , make them join a-C atoms via: set hbonds backbone , color the H-bonds using the scheme on the handout p10 via: color hbonds type . Some inter-mainchain H-bonded motifs appear, as on the handout p1-8. Make into a movie, with 360º rotation, as on the handout p16. Insert into a Powerpoint document.

45. P6 Ser/thr OH binding to main chain CO 3 or 4 residues behind. A common feature in the middle part of a-helices, especially in TM regions, where they may modulate helix bending. Deupi et al 1994 Biophys J86, 105.

46. An asx-motif (asn). Common at the N-termini of a-helices.