proteinsSTRUCTURE O FUNCTION O BIOINFORMATICS

Evidence for novel quantum chemistry toform triple and quadruple cysteine bridgesJochem M.G. Evers, Wouter G. Touw, and Gert Vriend*

Centre for Molecular and Biomolecular Informatics, Radboud university medical center, Geert Grooteplein-Zuid 26-28, 6525 GA, Nijmegen, The Netherlands

ABSTRACT

The question why cysteine bridges always contain only two cysteines has been raised many times because energy calculations

strongly suggest that triple and quadruple cysteine bridges can also exist. The unprecedented growth of the PDB has stimu-

lated us to once again search for these elusive products of protein structure evolution. And this time we were successful. In

total, 36 triple cysteine bridges in 18 different PDB entries, and 30 quadruple cysteine bridge in 5 different PDB entries

were observed. On top of that 6 cysteine structures in which all four cysteines are bridged to each other were found in 6

different PDB entries. Our excitement got combined with amazement when we realized that in all cases we observed a zinc

atom at a short distance of the sulphur atoms. A thorough quantum chemical analysis revealed that zinc ions can catalyse

cysteine bridge formation. A modelling study suggests that cysteine bridges consisting of five cysteines can also exist, and

we believe that it is only a matter of time before we will observe this intriguing structure element in a PDB file.

VC 2015 Wiley Periodicals, Inc.

INTRODUCTION

A PubMed search reveals only six articles with the

term ‘cysteine bridge’ in the title, the oldest of these by

Ross, Zhang and Selman (1995). This is a bit surprising

as the oldest recorded cysteine bridge in the PDB (2cha)

dates from 1972 (Birktoft and Blow, 1972). Clearly, the

importance of cysteine bridges has long been underesti-

mated, which can be explained only from our limited

understanding of their path of formation. Studies on cys-

teines have been published as early as 1981 (Thornton,

1981). Many researchers have attempted to study cysteine

bridge formation (e.g. Weissman and Kim, 1995; Darby

et al., 1995), but this topic has been marred in contro-

versy, and we believe that the underlying problem has

been the incomplete understanding of the most impor-

tant process in the process: zinc-catalysed sulphur-sul-

phur binding. This idea gets further corroborated by the

observation that the PDB contains almost 2500 entries in

which a zinc ion (Zn21) is observed in close proximity

to the Sg atom of one or more cysteines.

Although Zn21 is considered to be redox-inert in bio-

logical systems (Laitaoja, Valjakka and J€anis, 2013), Zn21

can be involved in redox reactions when coordinated by

redox-active cysteines (KreR _zel, Hao and Maret, 2007).

Moreover, zinc-coordinating cysteines form disulphide

bonds upon dissociation of Zn21 from Metallothionein

(Babu et al., 2014). Experimental evidence for the exis-

tence of a general mechanism of zinc-catalysed sulphur-

sulphur binding was not reported, though, in a recent

survey of zinc coordination in protein structures (Lai-

taoja, Valjakka and J€anis, 2013)

Here we report the property of cysteines to form more

than one cysteine bridge in the presence of Zn21. This

poly-bridging can reach up to three cysteine bridges per

sulphur atom, leading to an entire cysteine cluster.

RESULTS

The Protein Data Bank (PDB; Berman et al., 2000;

Berman et al., 2003; Gutmanas et al., 2014) was searched

for X-ray structures that contain zinc (�6000 entries)

and a subset was generated in which the zinc was located

close to a cysteine (�2500 entries). All representative

cases were visually inspected. Figure F11 shows a typical

case of a zinc – cysteine couple.

*gerrit.vriend@radboudumc.nl

DISCLAIMER: The following article is a special online-only Virtual Issue contri-

bution and it did not go through the Proteins: Structure, Function, and Bioinfor-

matics peer-review process.

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The location of the zinc close to the cysteine Sg in

Figure 1 can easily be explained using standard quantum

chemistry software. In Zn�Cys4 complexes, all four Sg

atoms normally are deprotonated and thus negatively

charged (Dudev and Lim, 2002). A quantum dynamics

simulation reveals the rapid equilibrium for the Cys clos-

est to the zinc:

COLOR

Figure 1PDB code: 2zp8, chain E, zinc 54. The Sg atom of Cys 12 (left) islocated 1.63 A away from the zinc ion (gray sphere). The other cys-

teines are located at a much bigger distance from the zinc than the“ideal” distance of 2.340 6 0.020 A according the REFMAC dictionary

(Vagin, 2004). The distances are 3.04 A, 2.89 A and 3.79 A for Cys 15Sg (top right), Cys 26 Sg (middle right) and Cys 29 Sg (bottom right)

respectively. Note that the zinc ion is shown at 30% of its Van der

Waals radius. The 2mFo-DFc map and mFo-DFc map are contoured at1.5 r (blue mesh) and at 1 and - 3 r (green and red meshes), respec-

tively. The electron density map sampling rate is a third of theresolution.

COLOR

Figure 2(A) PDB code 1r9s, chain B, zinc 1307. The Zn21-Sg- 1 Sg- situation where the distance to Zn21 is 1.94 A for Cys 1166 Sg (left) and 2.93 A forCys 1185 Sg (center). The cysteines are about to form a disulphide bridge. (B) PDB code 3gtk, chain I, zinc 204. The Zn21-Sg–Sg- situation where

Cys 103 (top, Sg 1.71 A from Zn21) and Cys 106 (left, Sg 2.08 A from Zn1) have just formed a disulphide bridge. C) PDB code 3h0g, chain N,

zinc 2225. The Zn21 1 Sg-Sg situation where the Sg atoms from Cys 1155 (top left) and Cys 1173 (bottom left) are 2.16 A and 2.35 A away fromthe Zn21, respectively. The disulphide bridge is 2.04 A long.

COLOR

Figure 3(A) PDB code 1tug, chain D, zinc 155; linear triple cysteine 141 (topcentre). Ser 116 helps to coordinate the zinc, as well as the Cys 114 Sg

(at 0.79 A from the zinc ion) and Cb (at 1.99 A) atoms. The SSBONDrecords in PDB entries do not yet allow the definition of trisulphide

bonds. The authors solved this problem by specifying the two S-S

bonds in two separate records; each of the two SSBOND records con-taining Cys 141. (B) PDB code 4bn5, chain A, zinc 1397; fully con-

nected triple cysteine. (C) PDB code 1nlt, chain A, zinc 352; fortunatelycaptured intermediate. D) PDB code 3d7s, chain B, zinc 313; the final

product: the quadruple bridge.

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Evers et al.

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Zn211 Sg2 $ Zn212Sg2 (1)

The Zn21-Sg- species is reactive, and prefers to react

with lone-pairs on soft Lewis bases like those of sulphur or

nitrogen. We ran a quantum dynamics simulation on the

situation in which one sulphur is very close to the Zn21

and another reduced cysteine ready to form a disulphide

bridge (see FigureF2 2A). This simulation suggested that the

reaction as given in eq. 2, is highly likely to occur.

Zn212Sg21 Sg2 $ Zn212Sg–Sg2

$ Zn211Sg2Sg12e2 (2)

And indeed, each stage in this process has occurred in

some entries in the PDB (Figure 2). The negatively

charged Zn�Cys4 complexes tend to be screened by posi-

tively charged amino acids (Maynard and Covell, 2001).

These basic residues are most likely responsible for the

generation of hydrogen gas:

2NH3112e2 $ 2NH212H112e2 ! 2NH21H2 (3)

The protons may need to be bound temporarily to

any hydrogen acceptor. We assume that this is the main

reason that we so often observe histidine side-chains in

close proximity to zinc ions.

Equation 2 can easily be extrapolated to obtain chained

or even triangular cysteine bridges. However, as the reac-

tion releases hydrogen gas, and as H3 doesn’t exist, we

believe that the final product in these cases must be a com-

plex bridge including four sulphurs from four cysteines.

The latter would then cause the release of the quantum

chemically much more sensible 2H2. A scan of the PDB

revealed something surprising (Figure F33).

In Figure 3C we see the final intermediate, the Zn21-

Sg–Sg–Sg–Sg- situation. Surprisingly, the four sulphurs

bind around the zinc catalyst (fig 3C). This means that

the zinc must tunnel out of this cage to reach the final

situation as observed in Figure 3D.

A quantum dynamics simulation starting from the five

cysteines around Zn-607 in 2pvc chain C (see Figure F44)

teaches us that cysteine bridges consisting of five cys-

teines must exist. We could however not yet find experi-

mental evidence for such a structure element in the PDB.

DISCUSSION

A famous Californian bioinformatician once said that

“when it comes to protein structure geometry, there

COLOR

Figure 4Five cysteines around zinc. PDB code 2pvc, chain C, zinc 607.

COLOR

Figure 5Zn 155 D in 1tug. (A) PDB structure. (B) Manually corrected and re-refined structure. Maps as in Figure 1.

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must be room for freedom of interpretation”. We want

to issue a stern warning against this idea, after all, who

can argue against quantum chemistry? Too long have

we taken the liberty of freely interpreting the data

(reflections) and freely choosing the constraints upon

refining protein crystal structures. This can lead to big

anomalies as we determined by comparing the struc-

tures mentioned in this study with their PDB_REDO

(Joosten, Salzemann et al., 2009; Joosten, Womack

et al., 2009; Joosten et al., 2012) mates. The structure

shown in Figure 2A, for example, suffers from the lack

of freedom of interpretation because upon re-

refinement with Refmac (Murshudov et al., 2011) it

gets forced into a dull, featureless and highly symmetric

tetrahedron as shown in FigureF5 5B. The manual re-

interpretation and re-refinement of the experimental

data for PDB entry 1tug (Figure 3A) suggested an

alternative mechanism of zinc coordination in aspartate

transcarbamoylase that does not involve Ser 116 (Figure

5). We prefer the original interpretation, however, since

the conformation of the tetrahedron we obtained is too

good to be true.

We believe that this article beautifully illustrates the

power of the PDB, a power that mainly comes from its

numbers. The large quantity of files of proteins solved at

increasingly high resolution allows us to better separate

the rules from the exceptions and the interpretations

from the miss-interpretations. And at times, especially

around early April, this can lead to amazing new

discoveries.

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