James Milner-White

James Milner-White

Glasgow University, UK

[email protected]

In -helix, -sheet and polyproline II helixthe main chain conformations of successiveresidues are identical. But if they are not?

Small Motifs (up to 8 residues)

-turns 19% motifs 14%nests 7%asx-, ST- features 7%Schellmann loops 7%-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

small, hydrogen bonded, motifs that recur in proteinsP2-7

Right- and left-handed forms

All main chain motifs can occur inenantiomeric forms.e.g. : RH and LH -helix.

Interconvert them by multiplying and by –1.

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

H HHHHHHHH H O O OO OO O O O O

other =

Main Chain – Main Chain Hydrogen Bond Color key

R 2 3 4 5-3-4

CO binds to NH R residues ahead

Invoke this color scheme in Rasmol by typing: color hbond type

P10

-turns 19% motifs 14%nests 7%asx-, ST- features 7%Schellmann loops 7%-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

small, hydrogen bonded, motifs that recur in proteinsP2-7

The Schellmann loop

occurs at the C-terminus of a third of all-helices

M-W 1988 JMB 199, 503

P5

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 JMB 315, 171

The NestP2

Carbonyl oxygen atoms are anionic in character

C = O-+

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

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 JMB 315, 171

The NestP2

Two Nest Conformations

Nests have two types of characteristic angles for

residues 1 and 2. The angles for residue 3 do not

affect nest conformation.

R, followed by L = RL

L, followed by R = LR

RL and LR are enantiomers, regarding their main chain atoms.

Average : -94, -1 77, 21 (80%)

Average : 77, 12 -82, -11 (20%)

P11

Left-handed-helix

Right-handed-helix

Nest R

Nest L

Enantiomeric conformations

180

0

-180

1800-180

Positive value conformations are more comfortable as glycines,

Genetically encoded amino acids apart from glycine prefer negative values.

P11

RL nests occur in

all Schellmann loops

The nest in the Schellman loopat the C-terminus of an -helix.

LR nests occur in …

The oxyanion hole of trypsin-likeserine proteases

CC

O

N

H

C

O

195

197

195

NH

H HN N

Trypsin- PTI

oxyanion holeof trypsin

‘Substrate’ of PTI

Peptide bond to be cleaved

195

197

An LR Nest occurs in Vancomycin

Is a complex glycopeptide antibiotic, with residues of alternatingD and L configurations. It acts by binding to a C-terminal D-alaresidue, an intermediate in bacterial cell wall synthesis. In thecrystal structure of the vancomycin-acetate complex, acetatemimics the terminal D-ala carboxylate.

P11

Vancomycin-acetate complex

Vancomycin: spacefill

Acetate: sticks

The three bluenitrogen atomsof the LR nest bindthe carboxylate oxygen atoms

Compound nests

As well as simple nests, alternating RLR, RLRL,or LRL, etc., sequences occur. In thesethe NH groups all face the anion, resultingin a wider, compound, nest that tendsto bind anionic groups rather than atoms.

Functionally Important Nests and Compound Nests Nest type Resno PDBcode protein Anion Ligand 

LR 195 trypsin O peptideLRLR 13 5p21 ras P GTPLRLR 48 1aqu estrogen sulfotransferase P pyridoxal phosphateRLR 12 1rcf flavodoxin P FMNRLR 88 1amo NADPH cyt p450 reductase P FMNRL 142 1rie Rieske iron-sulfur protein I Fe2S2RLRLR 39 1a70 spinach ferredoxin I Fe2S2RLRLR 9 2fdn Clostridium ferredoxin I Fe4S4RLRLRRLR 57 1qla fumarate reductase I Fe2S2RLLRLR 46 1cje adrenodoxin I Fe2S2RLR 387 1qj2 CO dehydrogenase molybdo-pterinRL 212 1az2 aldose reductase P NADPRLR 37 1rge ribonuclease O of guanine of rntdRLR 1bgs barnase P RNALRLR 179 2nmt N-myristoyl transferase P myristoyl-CoALRLR 119 1bo4 N-acetyl transferase P acetyl CoARLR 129 1opr orotate P.R.transferase P PRPPRL 741 RNA polymerase ’chain - -RLR* 6 1qfu Haemagglutinin -chain O D112LR* 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 acarbonyl or carboxylate oxygen atom (O), a phosphate group (P) or an iron-sulphur center (I).  

The P-loop

The ATP/GTP binding P-loop has an LRLR compound nest, bindingthe -phosphate of ATP or GTP.

PO

O

O

O

P

P

13

17

N

H

H

H

N

N N

NH

H10GAGGVGKS17

LRLRP21ras sequence

P12

P-loop of ras

-phosphateGTP oxygens

Five main chainNH groups

(Red)

(blue)

P12

Iron-Sulfur ProteinsThe longest compound nests (e.g.: RLRLR) surround iron-sulfur centres such as:

cubes

Fe

Fe

S

SFe

Fe

S

Ssquares

S

Fe Fe

S

These centres in proteins have a net negative charge when the extra cysteine sulphurs are considered, so are anions.

S SS

S

S

S

N

N

NN

N

NS

FeS

SFe

S

S S

H

H

H H

H

H39

44

Ferredoxin Fe4S4 (+S4) cube P12

ArchaealFerredoxinwith Fe3S4

Centre

N

O

H

Ca

OOO

N

N N

NH

HH

HO

O22

22

20

24 26

EF hand, from calmodulin

Three carboxylates bind the Ca++

A compound nest binds the carboxylates.

P13

22

24

20

EF hand fromcalmodulin

Ca++

Left-handed-helix L

Right-handed-helix R

Nest R

Nest L

180

0

-180

1800-180

P11

Potassium Channels

Passing from the RL to the RL conformationthe nest cavity becomes shallower; and thepolypeptide is more linear.

RL conformations are uncommoncompared to RL, with one exception.

Potassium channels are tetramers; the potassium ionspass through a channel formed by the CO groups of 4identical polypeptides from each subunit, with the RL conformation.

Watson& MW 2002 JMB 175 199

Morais-Cabral et al., 2001 Nature 414, 37-42 43-48 Potassium channel

Selectivity filter

Potassium=pink

The selectivityfilter of thepotassium channel

Potassium = pink

Single chain ofselectivity filterof potassiumchannel withpotassiums in place.

Potassium = pink

T

V

G

Y

P13

Each potassium in the channel is surrounded by eight carbonyl oxygens

P13

Catgrips

The nest (and K+ channel) conformations all haveapproximately alternating enantiomeric main chainresidues. Are there other conformations of this sort?

Yes. In polypeptides where alternate main chainCO groups bind Ca++ ions. We call them catgrips.

They have RL or LR conformations, RL or LR.

Average angles are: R –64, 132 L 78, -160

Like nests, they can be, and often are, compound.

Watson& MW 2002 JMB 175 199

Catgrips 

Catgrip type Resno PDBcode protein

Four successive mainchain carbonyl oxygens bound to 2 CalciumsoLoRoLo 352,361,370 1af0 MMP: SerralysinoLoRoLo 352,361,370 1kap MMP: alkaline protease

Three alternating mainchain carbonyl oxygens bound to 1 calciumoR LoR Lo 27 2ran annexinoR LoR Lo 29 1bp2 phospholipase A2 

oR LoL Ro 160 2usn MMP: stromelysinoR LoL Ro 177 1mmp MMP:matrilysinoR LoL Ro 177 1nfc MMP: fibroblast collagenaseoR LoL Ro 156 1bzs MMP: neutrophil collagenaseoR LoL Ro 195 1buv MMP: membrane-type collagenase Two alternating mainchain carbonyl oxygens bound to 1 calciumoR Lo 99,185,259 2ran annexinoR Lo 323 1fza fibrinogenoR Lo 160 1q1b MMP: gelatinase

oL Ro 87 2tec thermitaseoL Ro 79 1af4 subtilisin CarlsbergoL Ro 80 1mpt M-proteaseoL Ro 80 1scj subtilisin EoL Ro 80 1mee mesentericopeptidase 

C

O|

C|O

C|O

C|O

Ca

Ca

C|O

C|O

Ca

C|

C|O

C|O O

Ca

C|O

C|O

C|O

Alkaline protease

Baumann et al 1993 EMBO J 12, 3357

Part of the calcium bindingunit of alkaline protease

P14

Baumann et al 1993 EMBO J 12, 3357

Phospholipase A2

Exploring the conformations with alternating enantiomeric residues

P14

C

K

180

0

-1800-90

-90

90

B D

E

F

L

J

HG I

A

1 08

6

1 0

8

6

6

8

1 0

180

0

-1800-90

-90

90

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 forcyclic peptides byDeSantis, Morosetti& Rizzo, (1974) Macromolecules7, 52 .

Why should polypeptides with alternating enantiomericconformations occur? The situation is comparable to that studied by Ramachandran and others where successive residues (in -helix -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.

We have made a -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

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 -C atoms via: set hbonds backbone , color the H-bonds using the scheme on the handout p10 via: color hbonds type . Some inter-mainchainH-bonded motifs appear, as on the handout p1-8.

Make into a movie, with 360º rotation, as on thehandout p16. Insert into a Powerpoint document.

Ser/thr OH bindingto main chain CO3 or 4 residues behind.

A common feature inthe middle part of-helices, especiallyin TM regions, wherethey may modulatehelix bending.

P6

Deupi et al 1994 Biophys J 86, 105.

An asx-motif(asn).

Common at theN-termini of-helices.