Structure

Article

Multiscale Conformational Heterogeneityin Staphylococcal Protein A: Possible Determinantof Functional PlasticityLindsay N. Deis,1 Charles W. Pemble IV,2,3 Yang Qi,1 Andrew Hagarman,1,4 David C. Richardson,1 Jane S. Richardson,1

and Terrence G. Oas1,*1Department of Biochemistry, Duke University, Durham, NC 27710, USA2Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710, USA3Macromolecular Crystallography Center, Duke University, Durham, NC 27710, USA4Present address: KBI Biopharma, 1101 Hamlin Road, Durham, NC 27704, USA

*Correspondence: oas@duke.edu

http://dx.doi.org/10.1016/j.str.2014.08.014

SUMMARY

The Staphylococcus aureus virulence factor staphy-lococcal protein A (SpA) is a major contributorto bacterial evasion of the host immune system,through high-affinity binding to host proteinssuch as antibodies. SpA includes five small three-helix-bundle domains (E-D-A-B-C) separated byconservedflexible linkers.Prior attempts tocrystallizeindividual domains in the absenceof abindingpartnerhave apparently been unsuccessful. There have alsobeen no previous structures of tandem domains.Here we report the high-resolution crystal structuresof a singleCdomain, andof twoBdomainsconnectedby theconserved linker.Bothstructuresexhibit exten-sive multiscale conformational heterogeneity, whichrequired novel modeling protocols. Comparison ofdomain structures shows that helix1 orientation isespecially heterogeneous, coordinated with changesin side chain conformational networks and contactingprotein interfaces. This represents the kind of struc-tural plasticity that could enable SpA to bind multiplepartners.

INTRODUCTION

The structural plasticity conferred by conformational flexibility

has increasingly been recognized as a likely determinant of func-

tion. For example, multiscale heterogeneity in the calmodulin

central helix most likely helps it in binding >100 protein targets

(Wilson and Brunger, 2000), and a concerted motion seen in

both nuclear magnetic resonance (NMR) and crystal structures

of ubiquitin is proposed to underlie its functional plasticity of pro-

miscuous binding to many different proteins with high affinity

(Lange et al., 2008). However, flexibility is manifested in a variety

of ways, depending both on the protein itself and on how it is

observed. Flexibility is apparent in X-ray crystallography as elec-

tron-density inconsistent with a single molecular model—either

fully separated peaks or anisotropic density shapes showing

Structure 22, 1467–

fluctuation of atom groupings. In this work, we refer to alternative

conformations as conformational heterogeneity rather than flex-

ibility because the latter term implies motion on a relevant time

scale, which cannot be determined by crystallography. Many

phenomena contribute to conformational heterogeneity in crys-

tal structures, from diverse crystal contacts to functionally rele-

vant conformational fluctuations on a wide range of time and

size scales.

Like ubiquitin, staphylococcal protein A (SpA) exhibits broad

binding specificity with other proteins. This protein allows Staph-

ylococcus aureus to evade the innate and adaptive immune sys-

tems, making it a significant challenge to human health. Among

virulence factors responsible for S. aureus pathogenicity, SpA is

the best studied and arguably the most important. It is a highly

abundant 42kDa multi-domain cell-surface polypeptide with

two functionally distinct halves (Figure 1A). The C-terminal half

anchors SpA to the extracellular surface of the peptidoglycan

cell wall via the LPXTG motif (Schneewind et al., 1992) and is

likely disordered due to its low sequence complexity. In contrast,

the N-terminal half is a series of five stable protein-binding do-

mains (E-D-A-B-C). Recent studies establish that the conserved

sequence KADNKF forms a highly flexible linker between all

domains except E to D, which uses the longer sequence

KADAQQNKF, also likely to be highly flexible (A.H. and T.G.O,

unpublished data). The five domains have sequence identities

of 74% to 91% (relative to A domain; Figure S1 available online)

and share the same three-helix-bundle topology. The folding of

each domain is thermodynamically uncoupled to the others

and displays a gradient of increasing stability toward the more

C-terminal modules (A.H. and T.G.O, unpublished data). In addi-

tion, the B domain rapidly unfolds and refolds approximately 70

times per second (Myers and Oas, 2001), and recent studies

establish the same property for the other four domains (A.H.

and T.G.O, unpublished data). All five domains can bind the Fcand Fab regions of host antibodies (Jansson et al., 1998), tumor

necrosis factor alpha receptor 1 (Gomez et al., 2004), von Wille-

brand factor (Hartleib et al., 2000), and the C1qR component of

complement (Nguyen et al., 2000).

To date, only two crystal structures of SpA domains have

been solved, both single domains in antibody complexes: B

domain with Fc (Protein Data Bank [PDB] 1FC2) (Deisenhofer,

1981) and D domain with Fab (1DEE) (Graille et al., 2000). These

1477, October 7, 2014 ª2014 Elsevier Ltd All rights reserved 1467

Hlx1Hlx2

Domain2

Hlx3

Linker

Cell wall attachmentE D BA C

Protein-binding domains

Conserved, highly flexible linkerDomain stability

*

X: CN Y: CN

Linker

C

N

Domain1

A

B C

Figure 1. Staphylococcal Protein A and the Crystal Structures of C

and B-B Domains

(A) Schematic showing the organization of SpA and its five protein-binding

domains. The conserved linker (green) between the E and D domains has a

three-residue insertion, indicated by a star.

(B) The asymmetric unit of C domain (cyan). The three helices are labeled

(Hlx1-3) and the conserved linker regions are indicated (arrow) and colored

dark blue.

(C) The asymmetric unit of B-B domain. The two copies of B-B domain are

colored in shades of green or purple. Each domain (Domain1 and Domain2)

and the linker in between are depicted as a ribbon drawing, where Domain2 is

rotated by 180�.See also Figure S1.

Structure

Conformational Heterogeneity of Protein A

structures show partner interactions with SpA, but they lack

comparison with unbound domain structures, and their lower

resolution (2.7–2.8 A) does not allow determination of multiple

conformations. The B domain/Fc cocrystal structure (1FC2)

lacks coordinates for most of helix3, which originally stimulated

some interest in the possibility that helix3 unwinds upon Fcbinding. However, subsequent NMR-based amide hydrogen

exchange and circular dichroism studies suggested that helix3

is indeed formed in the complex and that the lack of density in

this region of the 1FC2 data resulted from a crystallization arti-

fact (Gouda et al., 1998; Jendeberg et al., 1996; Torigoe et al.,

1990). Another issue discussed by the groups that determined

NMR solution structures of B domain and its variants is the

interhelical angle between helix1 and helices2 and 3 (Gouda

et al., 1992; Tashiro et al., 1997; Zheng et al., 2004). The latter

helices are nearly perfectly antiparallel in all structures, but

there is significant variation in the angle between helix1 and

helix2, depending on the structure. A low helix1-2 angle was

observed in the Fc complex (Deisenhofer, 1981), whereas a

larger angle was found in the original solution structures (Gouda

et al., 1992; Jendeberg et al., 1996), which were not constrained

by residual dipolar coupling data. These results led the authors

to propose that a conformational change in B domain takes

place upon Fc binding to give a low helix1-2 angle. This pro-

posal was supported by kinetic B domain/Fc binding studies,

indicating that a conformational change might contribute to

kon (Jendeberg et al., 1995). Two subsequent solution struc-

1468 Structure 22, 1467–1477, October 7, 2014 ª2014 Elsevier Ltd A

tures of Z domain (an engineered B domain variant) and E

domain, based in part on residual dipolar coupling constraints,

showed a lower helix1-2 angle in the absence of Fc, which

called into question this mechanism (Starovasnik et al., 1996;

Tashiro et al., 1997). However, the differences in helix1-2 angle

between most structures are not significantly larger than the

uncertainties in these most recent structures, so the existence

and possible role of a coordinated conformational change in

helix1, whose residues form the majority of the contacts with

Fc, remains unresolved.

Conformational heterogeneity is traditionally evaluated at two

scales: side-chain-rotamer conformations and tertiary-structure

rearrangements, such as hinge motions and secondary-struc-

ture reorientations. It includes local backbone differences such

as peptide flips or backrubs that accompany side-chain rotamer

sampling (Davis et al., 2006), small translational shifts of a few

residues, and also side chain changes between distinct rotamer

states, which involve large movements and significant energy

barriers. Large-scale heterogeneity involves concerted posi-

tional differences of many residues, including changes in helix-

helix positioning (Zheng et al., 2004) or b sheet twist and

rare cases of major refolding (Skehel and Wiley, 2000), as well

as the long-recognized interdomain hinge motions. Any of these

types of conformational dynamics can be integral to enzyme

function, allosteric regulation, induced-fit binding and functional

plasticity, by enabling the alternative structures required for

biological function.

To investigate the molecular basis for SpA flexibility and the

connection between local and global conformational heteroge-

neity more generally, we have determined several X-ray crystal

structures of SpA C domain and B-B (two B domains connected

by the conserved linker), all in the absence of any partner protein.

C domain was solved to 0.9 A resolution at cryogenic tem-

perature (Figure 1B). The structure shows many backbone and

side chain alternative conformations. Because previous work

suggested that cryogenic freezing limits protein conformational

heterogeneity (Fraser et al., 2011; Juers and Matthews, 2004),

we also determined a 1.4 A structure of C domain at room

temperature. The B-B construct, determined at 1.5 A resolution

(Figure 1C), is an informative mimic of the nearly identical A-B or

B-C pairs. Because the linker was fully visible, the B-B structure

reveals effects of the special case of linker proximity on confor-

mational heterogeneity. This work establishes that SpA protein-

binding domains exhibit extensive structural plasticity that

presumably helps enable a small domain to accommodate

multiple binding partners, and sheds light on the diverse nature

of that plasticity.

RESULTS

Overview of C Domain and B-B StructuresDiffraction data for P21 C-domain crystals were collected at

cryogenic and room temperatures and solved by single-wave-

length anomalous dispersion (Table 1). Both structures contain

onemolecule in the crystallographic asymmetric unit and include

all 58 residues of the SpA C domain (Figure 1B), plus a Zn2+ ion

that binds the chain ends. Both also share the same three-helix-

bundle topology seen in previous SpA-domain structures (Fig-

ure 1B). Including N- and C-caps (Richardson and Richardson,

ll rights reserved

Table 1. Data Collection and Refinement Statistics

C domain

(Cryogenic)

C domain

(Room

Temperature) B-B

Data Collection

Space group P21 P21 P65

Cell dimensions

a, b, c (A) 27.3, 38.4,

28.6

27.8, 38.6,

29.0

44.4, 44.4,

214.8

a, b, gg (�) 90.0, 117.4,

90.0

90.0, 118.2,

90.0

90.0, 90.0,

120.0

Wavelength 0.8 1.0 1.0

Resolution (A) 50.0–0.9

(0.92–0.9)a50.0–1.42

(1.44–1.42)a50.0–1.49

(1.52–1.49)a

Rsym 0.07 (0.20) 0.07 (0.33) 0.10 (0.62)

I/sI 33.7 (4.36) 29.7 (2.54) 28.0 (2.2)

Completeness (%) 97.5 (77.6) 97.0 (73.6) 99.9 (99.6)

Redundancy 6.1 (2.7) 5.6 (2.9) 6.8 (4.4)

FOM 0.84 0.75

Refinement

Resolution (A) 25.39–0.90 25.5–1.42 37.9–1.49

No. reflections 37,938 9,889 39,026

Rwork/Rfree 11.3/13.0 11.4/14.3 14.2/18.5

No. atoms

Protein 1,646 1,524 6,240

Zinc 2 1

Acetate 3 3

Water 134 50 409

B-factors

Protein 7.4 16.4 19.8

Zinc 17.8 12.7 –

Acetate 7.3 16.4 –

Water 19.6 36.3 32.5

Root-mean-square deviations

Bond lengths (A) 0.013 0.013 0.003

Bond angles (�) 1.554 1.412 0.712

Model validationb

Ramachandran

outliers (%)

0 0 0

Ramachandran

favored (%)

98.2 99.0 99.0

Rotamer outliers (%) 0 0 0.5

C-beta outliers (%) 0 0 0

Clashscore 0.00 0.00 0.00

Overall score 0.50 0.50 0.50aData were collected from a single crystal. Values in parentheses are for

the highest-resolution shell.bFrom the MolProbity-style validation in Phenix.

Structure

Conformational Heterogeneity of Protein A

1988), the residue ranges are helix1: 6–19, helix2: 23–37, and

helix3: 40–56.

Diffraction data for P65 B-B crystals were collected at cryo-

genic temperature and solved by molecular replacement using

cryogenic C domain as the search model (Table 1). There are 5

Structure 22, 1467–

sequence differences between B and C domains, plus an

F13W substitution in each domain of the B-B construct (Fig-

ure S1) to allow fluorescence detection (see Experimental Proce-

dures). Crystals of B-B contain two molecules in the asymmetric

unit (Figure 1C). To limit the confusion associated with having

two domains, two chains and alternative conformations, we

labeled the chains X and Y, the alternative conformations a–d,

and the residues as 1–58 for domain1 and 101–158 for domain2.

The overall conformations of chains X and Y of B-B match

closely, but domain1 and domain2 differ significantly, as does

the detailed heterogeneity. We define the interdomain linker

in B-B as the residues between helix3 of domain1 and helix1 of

domain2 (i.e., 58 and 101–105). In each chain, this linker is in

an extended conformation making substantial contact with

domain2 across the same fairly hydrophobic helix1-2 face that

binds to Fc. Because of this linker conformation, the two domains

are translated and flipped in orientation relative to one another,

forming a hook-like structure overall (Figure 1C.)

Overview of SpA Conformational HeterogeneityThe high-resolution data show that SpA domains have much

more conformational heterogeneity than observed in the previ-

ous crystal structures, or even than other crystal structures

generally. Discrete backbone alternative conformations were

identified for 55%of residues (31 of the 58 total) in room-temper-

ature C domain, 62% in cryogenic C domain, 61% in B-B chain

X, and 74% in B-B chain Y. Doing justice to such complexity

required non-traditional approaches to achieve consistency

within and between the alternative models, as described in the

Experimental Procedures section. The extensive alternative-

conformation modeling contributed to achieving the outstanding

validation statistics reported in Table 1.

In addition to side chain shifts riding on the more global back-

bone shifts, all three structures have extensive individual side

chain heterogeneity. Figure 2A shows examples for two sur-

face-charged side chains and an interior aliphatic side chain,

with electron density to support as many as four distinct

rotamers. To quantify conformational heterogeneity among all

six domains, we computed the maximum distance between

equivalent atoms of alternative conformations within each resi-

due along the sequence, separately for backbone atoms and for

side chain atoms. The 3D distribution of this heterogeneity

within single domains is shown for cryogenic C in Figure 2B

as the local width in a putty-sausage diagram. The greatest

side chain heterogeneity occurs at side chains pointing away

from the interior, not unexpectedly. This surface heterogeneity

occurs in side chains without strong intermolecular interactions,

but an exception is the Tyr114 side chains in B-B chains X

and Y, which interact with each other across molecules in the

asymmetric unit.

Figures 2C, S3, and S4 use various representations to

compare the distribution of conformational heterogeneity be-

tween the six different domains in these structures. The overall

pattern is very similar in the C domains and the first domains of

B-B. An interesting exception is the lower heterogeneity of C

domain at the beginning of helix3, close to the main cluster

of sequence differences between C and B. The most conspic-

uous contrast, however, is between domain2 of both B-B

chains and that of the other four domains. In particular, helix1

1477, October 7, 2014 ª2014 Elsevier Ltd All rights reserved 1469

C

Hlx1

Hlx2Hlx3

N

BackboneSidechainBoth

90°

Lys42Glu24

Ile346.29Å 3.74Å

1.03Å

5 10 15 20 25 30 35 40 45 50 55

Maximum Equivalent Distance (Å)Helix 1 Helix 2 Helix 3

0 1 2 3 4 5 6 7 8 9

Sidechain Alternate Conformations

Backbone Alternate Conformations

C RTC cryoB-B X1B-B Y1B-B X2B-B Y2

Residue No.

C RTC cryoB-B X1B-B Y1B-B X2B-B Y2

A

B

C

Figure 2. Residue-Level Conformational

Heterogeneity

(A) Examples of heterogeneity in C domain. Lys42

and Glu24 sidechains are solvent-exposed, and

Ile34 is in the interior. The maximum distance

between equivalent side chain atoms (see text) is

depicted for each residue.

(B) Putty-sausage diagram of C domain where

the relative diameter represents the maximum

distance between equivalent atoms of alternative

conformations (if present) within each residue. The

color coding reflects the type of heterogeneity.

(C) Comparison of residue-level conformational

heterogeneity of single domains in C domain and

B-B structures. B-B chain and domain designa-

tions are indicated as a suffix to the protein name.

For example, ‘‘X1’’ indicates domain1 of chain X.

See also Figures S3 and S4.

Structure

Conformational Heterogeneity of Protein A

is significantly less heterogeneous in domain2, probably due

to interaction between the interdomain linker and helix1 of

domain2 (discussed below). This perturbation by the linker

appears to induce greater changes in conformational heteroge-

neity than data collection temperature, sequence differences

between B and C domains, or the fact that C domain is 0.5 ±

0.15 kcal/mol more stable than B domain (A.H. and T.G.O,

unpublished data).

Conformational Coordination of Side Chain AlternativeConformationsMany residue-level conformational changes involve rotamer

shifts of neighboring side chains (Figures 3A–3C), where only

some local combinations of conformations are possible without

prohibitive steric clashes. Because electron density only shows

the sum of all conformations, other information is needed to

correctly analyze the conformational clustering, including all-

atom sterics and the logic of assigning alternative conformation

labels and occupancies.

The two domains within each B-B chain have nearly the same

backbone conformation, but differ in backbone heterogeneity

(see above) and also show differences in side chain conforma-

1470 Structure 22, 1467–1477, October 7, 2014 ª2014 Elsevier Ltd All rights reserved

tion and heterogeneity, some of which

occur in coordinated networks. An

extreme example is Tyr114 (domain2),

which adopts two distinct rotamer con-

formations, whereas Tyr14 (domain1)

adopts only one rotamer. Trp13 points to-

ward both Tyr14 and Leu17, which must

therefore both point away from Trp13

(Figure 3D), but Trp113 points away

from Tyr114 and Leu117, which lets

them adopt alternative conformations

(Figure 3D). The Trp13 conformation

in domain1 is incompatible with the con-

formations of Tyr114 and Leu117 in

domain2 because that combination

would produce serious steric clashes.

The position of Trp13 therefore con-

strains the conformations of surrounding

residues, whereas Trp113 is in turn limited by the presence of

the linker bound to domain2.

Based on refined occupancies of Gln110 in chain Y and Tyr114

in both chains, we can estimate the populations of three sets of

rotamers, which are depicted in Figures 3A–3C. The most popu-

lated set (Figure 3A) has a population of �60%, and the second

set (Figure 3C) has a population of �30%. The least populated

set (Figure 3B) has amuch lower population of�8%, presumably

because of a somewhat unfavorable contact between the Tyr

rings, which may move apart by an amount too small to be

distinguished within the summed density.

Heterogeneity in Helix1 OrientationWith the structures of unbound C domain and B-B solved, we

now have six structures of SpA domains with which to analyze

and compare conformational heterogeneity, as well as the

various single-domain structures already in the PDB. Montelione

and coworkers (Zheng et al., 2004) previously reported wide

disagreement over the tilt angle of helix1. Our structures demon-

strate that these differences represent actual differences in

helix1 orientation between structures, rather than errors in one

or more of the NMR structures. Our results confirm that helix2

Gln110-Yalt-a

Tyr114-Yalt-a

Gln110-Yalt-b

Tyr114-Yalt-a

Gln110-Yalt-b

Tyr114-Yalt-b

Tyr14

Trp13

Leu17

Tyr114

Trp113

Leu117

Tyr114

Trp13

Leu117

Tyr114-Xalt-a

Tyr114-Xalt-b

Tyr114-Xalt-b60% 8% 32%

Domain1 Domain2 Domain1,2

A B C

D E F

Figure 3. Concerted Conformational Het-

erogeneity in B-B

(A–C) Populations of Tyr114-X, Gln110-Y, and

Tyr114-Y for the alt-a (A, 60%), mixed alt-a/alt-b

(B, 8%), and alt-b (C, 32%) conformations.

(D–F) Trp13 in Domain1 (D) and Trp113 in Domain2

(E) constrain the rotamers of nearby residues,

whereas the mixed conformations of the two do-

mains when superimposed (F) are incompatible

with one another due to significant overlap of the

van der Waals spheres, as indicated by red spikes.

Structure

Conformational Heterogeneity of Protein A

is the least variable and helix1 the most, with more change

parallel than perpendicular to the helix2-3 plane. To allow

quantitative comparison of helix1 orientations among current

SpA-domain structures, we transformed the helix axis vectors

for each domain into the same coordinate system with helix2

as the principal reference (see Experimental Procedures). The

superimposed helix axes and the relative helix1 orientations for

ten different SpA-domain structures are shown in Figure 4A

and Table S1, respectively. Our crystal structures differ by

up to 7.5� from one another (Figure 4b), 10� from 1Q2N (Zheng

et al., 2004), and 13� from 1DEE (Graille et al., 2000).

Although there are some sequence differences between the

different domains, these nonconserved residues are not at the

helical interfaces, so differences in interhelical angles cannot

be attributed directly to local sequence differences. In addition,

nearly the full range is seen among B-B domains.We hypothesize

that subtle differences in packing due to residue-level conforma-

tional differences in and out of the direct interface are responsible

forhelix1 rearrangements insingle-domainSpAstructures.To test

this, we compared the interhelical packing in C domain with the

canonical B-domain NMR structure, 1Q2N (Zheng et al., 2004;

Figure 4C). Helix1 differs most toward its C-terminal end, with a

pivot point around Ile16. In analogy to Crick’s ‘‘knobs and holes’’

(Crick, 1953), this important ‘‘knob’’ side chain nestles into a deep

cavity between helix2 and helix3 and makes six side chain con-

tacts across the interface (Figure 4D). Ile16 adopts the same

conformation in both structures, and four of those six interface

contacts (to Phe30, Ile31, Leu45, and Ala48) are identical. This is

consistent with previous work (Braisted and Wells, 1996), which

suggested that Ile16 stabilizes the packing of helix1 to helix2.

The interaction of Leu45with Ala12 is also the same in both struc-

tures. Figure 4D shows the key knob residues on theN- andC-ter-

minal ends of helix1 that intercalate with ‘‘hole’’ residues across

the interface, thereby holding the three-helix-bundle together.

Most assume unique conformations in each structure, leading

to differences in knob locations (Figure 4D) when viewed down

the helical axis. For example, both Leu19 and Asp52 sidechains

shift significantly to maintain their contact, while Leu22 changes

rotamer from mt (minus, trans) to tp (trans, plus) in most 1Q2N

models tomaintain contactwith Ile19 in the newhelix1orientation.

Structure 22, 1467–1477, October 7, 2014

Effect of theB-B Interdomain Linkeron Conformational HeterogeneityIn the context of the B-B structure

(see Figure 1C), both interdomain linkers

form a rather extensive and well-packed

interface with helices1 and 2 of domain2

(including Phe105 and Trp113), on the same surface region

that binds Fc in the 1FC2 complex structure. Recent NMR

studies established that six residues (K58A101D102N103K104F105)

within the linker sequence are highly flexible in solution (A.H.

and T.G.O, unpublished data). For this reason, we know that

no single domain-domain arrangement, including this one, can

predominate in solution. Given the linker flexibility, this inter-

action must be quite weak, yet it seems the most likely cause

of the substantial reduction in conformational heterogeneity for

helix1 of domain2 in both B-B chains (Figure 2C). This conforma-

tional sensitivity of helix1 to contacts with other parts of the

molecule suggests that its orientation can be readily frozen out

when it is part of an interface.

Relationship of Conformational Heterogeneity toDiversity of Binding PartnersThe large set of SpA-domain crystal structures now available,

especially the ones at high resolution, allows analysis of the

relationship between intradomain conformational heterogeneity

and the potential variety of binding partners. To do this, we

have focused on comparing the helix conformation and interface

packing of the 1FC2 B domain/Fc complex (Deisenhofer, 1981)

with the variety of conformations and interfaces seen in the other

crystal structures.

Table S1 shows that the helix1-2 interhelical angle is largest

for the two C domain and B-B chain Y domain2 structures

and very small for the B domain/Fc structure (1FC2). Therefore,

for maximum contrast, we docked the 1FC2 complex onto C

domain by superimposing the 1FC2 B domain onto the cryo-

genic C structure. We then examined the noncognate interface

between C domain and the docked Fc. The well-fit interface of

the cognate interaction (Figure 5A) is replaced with a combina-

tion of huge steric clashes and non-interacting gaps (Figure 5B).

This interaction actually occurs (because the C domain is known

to bind Fc; Jansson et al., 1998); therefore, the complex must be

capable of relaxing to fit. An attempt to see whether purely side

chain shifts in the C domain could alleviate the clashes was

surprisingly successful, but necessitated coordinated rotamer

changes for an entire network of large side chains and failed at

adding positive interactions. The majority of side chain atoms

ª2014 Elsevier Ltd All rights reserved 1471

1Q2NC domain

Hlx1

Hlx2

Hlx3

Pro38Lys35 Ser41

Arg27Asn52

Lys49Hlx2Hlx3

Hlx1

Gln9

Ile16

Leu19Hlx2 Hlx3

Hlx1

Lys35

Gln9

Asn52Arg27

Lys49

Leu19

Z Cryo C

A B C

D E

Figure 4. Global Conformational Heteroge-

neity of Hlx1 in SpAProtein BindingDomains

(A) Helix-axis orientations of sixteen different SpA

domains in a coordinate system where the x axis is

parallel to Hlx2, and Hlx3 lies nearly in the x-y

plane. The cryogenic C domain (C) and 1Q2N Z

domain (Z) structures are labeled.

(B) Superposition of both C domain structures with

the four individual domains of our B-B structure.

(C) Superposition of Z domain (1Q2N, variant of B

domain) and cryogenic C domain using Hlx2 and

Hlx3 for the superposition.

(D) Peel-away of interior of the Hlx1-Hlx2/3 inter-

face, shown from both sides. Interfacial sidechains

are highlighted with colored spheres. The key core

residue at the interface is Ile16, which forms the

pivot point for variation in the angle of Hlx1.

(E) The helix differences between 1Q2N and C

domain are the consequence of significant re-

arrangements in the interhelical knob packing for

all three helices. Residues and spheres are colored

as in (B), which represent structure affiliation. The

pivot, marked in the figure, consists of Ile16.

Structure

Conformational Heterogeneity of Protein A

consistently shifted toward the surface of the complex, leaving

gaps in the packing.

Measurement of interfacial solvent-accessible surface area

(Chothia, 1976) is a useful way to correlate structure to binding

affinity for evolved, well-packed interfaces, but ignores gaps of

up to 2.8 A. As a more sensitive measure for this modeled and

possibly ill-packed interface, we used all-atom contact analysis

(Word et al., 1999), which counts only atom-atom contacts within

0.5 A of ideal van der Waals distance. The cognate interface in

1FC2 has 142 A2 total all-atom contact, 110 A2 of favorable

H-bond plus van der Waals, and one clash (see Table S1 for de-

tails). The initial noncognate interface has only 54 A2 favorable

and 12 clashes; the rebuilt noncognate interface has only one

clash but still just 68 A2 of favorable contact. For comparison,

the internal interface between helix1 and 2-3 is 153 A2 total

(147 A2 favorable), the B-B linker contacts domain2 across

106 A2, and a sample of tight biological dimer and inhibitor inter-

faces ranged from 100 to 460 A2. Crystal contacts typically have

fairly little direct contact—a sample ranged from 18 to 40 A2. Of

note for SpA, however, C domain makes crystal contact across

121 A2 plus a Zn site, and the B-B crystals alternative contacts of

144 and 258 A2; presumably this helps attain high resolution.

The coordinated network of changes in side chain conforma-

tion found in the noncognate modeling is recapitulated else-

where in our crystal structures, and it gives a broader context

for the constraints on possible rotamer pairings seen in Figure 3

when assigning consistent alternative-conformation models in

the B-B structure. Of our six domain structures, the high interhe-

lix angle and side chain arrangement of theC domain is also seen

in the second domains of B-B, whereas the low interhelix angle

and side chain arrangement of 1FC2 B domain is seen in the first

domains of B-B. Notably, all six domains make extensive crystal

contacts across this helix1-2 surface, in three different arrange-

ments. As shown in Figures 5C and 5D, seven side chains make

concerted rotamer changes: Ile31 on helix2 and Gln10, Asn11,

1472 Structure 22, 1467–1477, October 7, 2014 ª2014 Elsevier Ltd A

Phe/Trp13, Tyr14, Leu17, and His18 on helix1. Some of the co-

ordination between adjacent side chain rotamers is constrained

but not all: the more exposed Asn11 and His18 adopt multiple

alternative conformations, and are not sequence conserved.

In summary, there is substantial coordination between backbone

and side chain conformational heterogeneity in SpA domains,

and a probable relationship between that combined hetero-

geneity and the ability to form contacts with a variety of protein

partners.

DISCUSSION

Interpreting Conformational Heterogeneity inCrystallographic DataRepresenting protein structures as collections of possible

models consistent with the data is common practice for struc-

tures determined by NMR. Although these are often called en-

sembles, there is usually no direct evidence that they represent

the true ensemble of structures present in solution. Recent ef-

forts to incorporate dynamic NMR information into NMR model

refinement have produced more realistic representations of

true ensembles (Lindorff-Larsen et al., 2005). Ensemble building

is a relatively new pursuit in X-ray crystallography, and three cur-

rent methodological approaches are promising but still in their

infancies. The first approach repeats crystallographic model

building and refinement from multiple randomly seeded starts

(DePristo et al., 2004; Terwilliger et al., 2007), which is good at

identifying uncertainty within the major conformation but seldom

captures large differences or minor populations. The second

approach is molecular dynamics-based ensemble refinement,

for instance as currently under development in the PHENIX crys-

tallographic suite (Adams et al., 2010; Burnley et al., 2012). When

we tried it on our structures, individual results failed validation

and only some of the side chain alternative conformations

evident in electron density were captured. A third approach is

ll rights reserved

Hlx2 Hlx1 Hlx2 Hlx1

Cognate:Low Angle High Angle

Fc Fc

Hlx2 Hlx2Hlx1 Hlx1

High AngleLow Angle

10

18

31

A B

C D

Figure 5. Coordination of Backbone and Side Chain Conformational

Changes with Binding of Fc(A and B) All-atom contacts at binding interfaces. (A) Well-fit, cognate interface

of Fc with SpA B domain (low-angle helix 1-2 orientation), in PDB file 1FC2; (B)

Poorly fit, noncognate interface of Fc with superimposed high-helix-angle

conformation of C domain from 4NPD.Green and blue dots showgood van der

Waals contact or H bonds, and clusters of red spikes show steric clashes.

(C and D) Correlation of changes in helix-helix angle with concerted rotamer

changes in a network of seven side chains (bold colors) for (C) two low helix-

angle structures (B-B domain 1 for both chains), similar to the B domain in

1FC2, and for (D) two high helix-angle structures (C domain and B-B chain Y

Domain2). Switching only side chain network conformations on the super-

imposed, high-angle, noncognate C model from (B) can eliminate most

clashes but neither recreates good van der Waals packing nor any H-bonds.

However, the high-angle domain structures each form an extensive, well-

packed contact with another SpA-domain partner in the crystal.

See also Table S1.

Structure

Conformational Heterogeneity of Protein A

the qFit program (van den Bedem et al., 2009), which uses low-

level electron density to identify potential alternative conforma-

tions for the next atom but has not yet been extended to model

complete alternative side chain conformations or to correlate

neighboring changes. Automated methods for modeling and

coupling conformational networks, like that used in qFit and

CONTACT (van den Bedem et al., 2013), respectively, speed

up the tedious process of modeling side chain heterogeneity

and very accurately couple side chain alternative conformations.

However, the subtle anisotropy observed in our electron density,

particularly that of the backbone, is difficult for the current

automatedmethods to fit. Therefore, we usedmanual placement

of sidechain and backbone atoms to identify all alternative

conformations.

Because of these difficulties, our initial steps toward building a

complete, consistent conformational ensemble from the crystal-

lographic data were limited to careful identification of alternative

conformations one residue at a time. To accomplish this, we

extended traditional modeling of discrete alternative conforma-

tions based on anisotropy or separate peaks in the electron

density (see Experimental Procedures). Briefly, we kept isotropic

B-factors until all visible backboneandsidechain alternative con-

formations had been built. These conformations were assigned

Structure 22, 1467–

to specific, internally consistent models of alternative conforma-

tions using a combination of MolProbity conformational and all-

atom clash analysis, plus PHENIX refinement of occupancies.

Origin of High SpA-Domain ConformationalHeterogeneityIncreasingly more structures in the PDB break 1 A resolution,

where alternative conformations are most visible. Even among

these ultra-high-resolution structures, however, most have

about 10% backbone heterogeneity; a few (e.g., 1EXR, 2I16,

1YK4, 3NIR, and 2VB1) include alternative conformations that

stretch for more than two residues and have between 25%

and 36% backbone alternative conformations. We have found

only the PDB ID 1M40 b-lactamase (Minasov et al., 2002), fit

with 63% backbone alternative conformations, in or near the

indeed unusual 55%–74% range seen in our SpA structures.

The backbone alternative conformations within a single SpA-

domain crystal structure differ by only 1 A or less, but their

pattern is very similar to the larger changes between structures.

Prevalence of side chain alternative conformations in SpA is

large but hard to compare quantitatively with other structures

because many ride on backbone shifts.

There are several features of SpA that may lead to this unusual

conformational heterogeneity. For example, SpA domains are

small, single-chain three-helix bundles, each held together by

the Ile16 pivot residue (Figure 4D). The lack of an extensive hy-

drophobic core might facilitate conformational heterogeneity

by allowing adjustment of interhelical angles with less constraint

from interior van der Waals interactions.

Another consideration is that multiple backbone conforma-

tions may be less rare in other proteins than suggested by their

occurrence in crystal structures, because all types of partial

disorder are more prevalent at low resolution but can be well

characterized only at high resolution. An unusual feature of

SpA domains may be that, despite having high conformational

heterogeneity, they can still form high-resolution crystals. All

our crystals have extensive contacts that form a continuous

zigzag of domains along a crystallographic screw axis—a heter-

ologous contact in P21 for C domain, and an alternation of

domain1 and domain2 pseudo-2-folds in P65 for B-B, each of

which uses the helix1-2 face. The overall domain orientations

must repeat in exactly 180� for P21 and in exactly 60� for P65,

which these domains achieve using varied interhelical angles

and side chain positions. Therefore, we speculate that these

well-ordered crystals are enabled by the same plasticity used

to bind different biological targets.

Another unusual property of SpA domains is that they unfold

and refold very rapidly, in less than 100 ms (Myers and Oas,

2001; A.H. and T.G.O, unpublished data). Although kinetic and

thermodynamic experiments establish that the folding reaction

is two-state, the rapid kinetics implies a relatively low activation

barrier between the folded and unfolded ensembles. This char-

acteristic of the overall energy landscape may be correlated

with the complex native-state landscape suggested by the

high conformational heterogeneity we observe. In any case, it

seems likely that evolution has converged for functional reasons

on a sequence that not only includes a flexible linker but also pro-

vides side chain networks that can support alternative helix

packing (Figure 4) and alternative binding interfaces (Figure 5).

1477, October 7, 2014 ª2014 Elsevier Ltd All rights reserved 1473

Tyr114alt-a

Tyr114alt-b

Leu432Arg2519

Asp136Asp36

Fab

Domain1 Domain2

Fc

Domain1

Domain2 Fab

Tyr14

Domain2 Domain1,2

A B

DC

Figure 6. Conformational Compatibility of B-B with Antibody

Binding

(A) Tyr114 alt-a (blue) adopts the same conformation as seen in the Fc/B

domain complex (1FC2, magenta), but Tyr114 alt-b (cyan) is incompatible. The

Tyr alt-a side chain is posed for interaction with Leu432 from Fc.

(B) The Asp36 conformation in Domain1 is able to bind Arg2519 of Fab (as it

does in the structure of Fab in complex with D domain, 1DEE), but the Asp136

conformation in Domain2 is not possible due to a steric clash with the Fabmolecule.

(C and D) Functional relevance of interdomain orientation. (C) The crystallo-

graphic interdomain conformation of B-B is incompatible with Fc binding

because when Domain1 is posed as in 1FC2, the second domain clashes with

the Fc molecule. To bind to Fc, B-B must adopt another interdomain confor-

mation. (D) The interdomain orientation of B-B is compatible with binding two

Fab molecules, when superimposed with 1DEE.

Structure

Conformational Heterogeneity of Protein A

Possible Functional Roles of IntradomainConformational HeterogeneityFrom the structure of B domain bound to Fc (PDB ID 1FC2),

Deisenhofer identified Tyr14 and Gln10 as residues of B domain

involved in binding by forming H-bonds to backbone oxygens

of Fc (Deisenhofer, 1981). Gln10 is highly heterogeneous and

Tyr114 shows two distinct conformations in our structures (Fig-

ure S3), as part of a network of side chain differences coordi-

nated with helix1 orientation (see Results). We superimposed

the main chain atoms of residues 110–118 from B-B domain2

on the same residues in 1FC2. Only Tyr114 alt-a (Figure 6A)

t90 is capable of forming the H-bonds for the Fc interface while

avoiding clashes, and only when on a low-angle helix1 confor-

mation. Tyr114 alt-b is not compatible with binding because it

sterically clashes with the Fc molecule.

Similarly, Silverman and coworkers implicated Asp36 as a

residue on D domain that binds antibody fragment Fab via

H-bonding to Arg2519 (Graille et al., 2000). In our set of

structures Asp36 and Asp136 are quite heterogeneous, using

different rotamers to form H-bonds with different partners. To

see which of these conformations are compatible with binding

Fab, we superimposed both domain1 and domain2 of B-B onto

1DEE (Figure 6B). Asp36 in domain1 shows even slightly better

H-bonds to the Arg than in 1DEE, whereas Asp136 in domain2

clashes seriously with the Fab.

1474 Structure 22, 1467–1477, October 7, 2014 ª2014 Elsevier Ltd A

Another intriguing side chain-level feature of the current struc-

tures is a possible role forPhe5 in binding interactions that involve

the helix1-2 surface. The Phe5 side chain flips out to allow con-

tacts with a secondmolecule in the 1FC2, 1DEE, and 1H0T com-

plexes. Given the rather low order parameter of its NH in solution

(A.H. and T.G.O, unpublished data), Phe5 must spend consider-

able time disordered, along with the rest of the linker. However, it

is tuckeddownover one endof the helix1-2 surface in all six of our

SpA-domain structures and in the NMR structures 1SS1, 1BDD,

2SPZ, and 1Q2N (where NOEs were observed between the Phe

ring and domain surface residues (Doreleijers et al., 2005)). The

helix1-2 surface, the most hydrophobic face on SpA domains,

forms three quite different, well-packed crystal interfaces in our

structures, and is likely to play an important role in binding other

biological partners of SpA besides Fc.

Most interestingly, the large total set of SpA domain structures

now available, especially the ones at high resolution, allow study

of possible coupling between intradomain conformational het-

erogeneity and the variety of binding partners. We have shown

that: (1) helix1 orientation differs by 7.5� among our six domain

structures, and up to 13� from other crystal and NMR structures;

(2) helix orientation is correlated with a network of side chain ro-

tamer changes on the helix1-2 face (Figure 5); and (3) Fc binding

to that face is compatible only with the low-helix-angle form of

the concerted side chain/helix rearrangement, whereas other

well-packed contacts incorporate the high-angle version (Table

S1). It is extremely difficult to establish that backbone conforma-

tional change is essential for binding many partners, but we can

definitively say that concerted backbone/side chain changes are

in fact used for that binding.

Multidomain Conformational Preferences in SpAThe contact between D domain and Fab uses a different face of

SpA domains than the contact between B domain and Fc. As a

result, a single domain could actually bind both Fab and Fc at

the same time, which Graille et al. (2000) demonstrated by build-

ing a hypothetical model of the ternary complex. Whereas some

side chain conformations and helix orientations found in C

domain and B-B are compatible with binding Fc (as discussed

above), the domain-domain relationship observed in each B-B

chain would block binding of Fc to either domain due to major

steric overlap with the other linked domain (Figure 6C). This

observation confirms the NMR evidence that, in solution, B-B

is free to adopt many other domain-domain conformations,

thus allowing Fc to bind. Unlike the case with Fc, the global

conformation of the B-B chain is compatible with binding up to

two Fab molecules simultaneously on the negatively charged

helix2-3 faces, with no steric interference (Figure 6D). Cocrystals

of B-B or other SpA multidomain constructs with Fc or Fab would

be desirable to understand SpA binding.

EXPERIMENTAL PROCEDURES

Plasmid Construction

The C-domain gene was PCR cloned from the SpA-N gene. The PCR primers

added a 50 NdeI and a 30 BamHI site, andwere subsequently cloned into the T7

expression plasmid pAED4 (Doering, 1992). The B-B gene was synthesized by

GENEWIZ in a pUC57 cloning vector, and was subsequently cloned into the

pAED4 expression vector. It contained the F13W substitution to aid detection

of the protein.

ll rights reserved

Structure

Conformational Heterogeneity of Protein A

Protein Expression and Purification

Plasmids were transformed into Escherichia coli BL21(DE3) cells using stan-

dard transformation procedure. A single colony of transformed bacteria was

used to inoculate a 50ml culture of Luria brothmedia with 0.1mg/ml ampicillin.

This starter culture was incubated at 37�C until the optical density (OD)

reached 0.8–1.0, whereupon it was used to inoculate 1 l cultures that were al-

lowed to grow toOD 0.8–1.2 at 37�C. Isopropyl-beta-D-thiogalactopyranosidewas then added to a final concentration of 1 mM, and the cultures were incu-

bated for an additional 6–8 hr. The cells were harvested by centrifugation and

resuspended in 20–30 ml of 50 mM Tris pH 8.8, 1 mM EDTA, and protease in-

hibitor cocktail (AEBSF, pepstatin, bestatin and E-64). The cells were lysed in a

French pressure cell, and insoluble material was centrifuged from the lysate.

The lysate was brought to pH 9.0, and micrococcal nuclease was added to

digest large DNA fragments for 15 min. The resulting solution was brought to

4 M guanidinium HCl (BioBasic) and 20 mM TCEP was added. In two succes-

sive steps, the solution was dialyzed in a 5% acetic acid buffer, which precip-

itated many cellular materials, but not expressed proteins. After centrifugation

of the insolublematerial, the resulting solution was allowed to dialyze overnight

into deionizedwater. The protein was further purified using two types of ion ex-

change chromatography. First, the protein was loaded onto a strong cation

exchanging, SP Sepharose (GE) column in 50 mM acetate buffer at pH 3.6.

The column was eluted with a NaCl gradient (typically 100 mM to 500 mM

gradient) in a volume of 600–800 ml and collected in 10 ml fractions monitored

by a UV detector (Bio-Rad) at 278 nm. The fractions comprising the protein

elution peak were checked for purity by SDS-PAGE. The purest fractions

were pooled and dialyzed against deionized water. Subsequently, the resulting

solution was loaded onto a weak anion exchanging DEAE Sephacel (GE)

column in 25 mM Tris (BioBasic) at pH 10.0 and eluted with an 800 ml NaCl

gradient (typically 0–250 mM gradient) into 10 ml fractions monitored by a

UV detector at 278 nm. The protein elution peak fractions were checked for

purity by SDS-PAGE. The purest fractions were pooled and dialyzed against

deionized water. The final solution was lyophilized and stored in a desiccator.

Purities of the final protein stocks were confirmed by SDS-PAGE to be >95%

pure. The masses of all proteins were confirmed with electrospray ionization

mass spectroscopy.

Crystallization, Data Collection, and Initial Models

C-domain protein solution was prepared at 20 mg/ml from lyophilized protein

and mixed in a 1:1 ratio with crystallization solution (55 mM ZnCl2, 16.5% PEG

6000, and 0.1 M MES pH 6). Crystals formed by hanging-drop vapor diffusion

within five days and were of the space group P4332, although not of diffraction

quality. Seed stock was prepared by adding approximately 10 of the P4332

crystals into a 50 ml aliquot of well solution in a Hampton Seed Bead, and

the resulting microseeds were used in a new crystal screen. Diffraction-quality

crystals were formed by hanging-drop vapor diffuction within 2 days by mixing

C domain, well solution (30 mM NaSCN, 26% [w/v] PEG 3,350, and 0.5% [v/v]

glycerol), and seeds in a ratio of 3:2:1, respectively.

For the cryogenic data set, cryopreservation of the crystal was achieved

with the addition of 30% glycerol to the crystallization condition, and the crys-

tal was frozen by direct immersion in liquid nitrogen. All data were collected

remotely at the Advanced Photon Source (APS) at Argonne National Labora-

tory, beamline 22-ID (SER-CAT). Data were processed and scaled with

HKL2000 (Otwinowski and Minor, 1997). For the cryogenic C-domain struc-

ture, despite an I/sI of greater than 2 at the next higher resolution bin, the

data were truncated to 0.9 A resolution because completeness dropped below

70%. C-domain structures were solved using the single-anomalous dispersion

method at the high-remote for zinc at 0.8 A wavelength (cryogenic structure)

and 1.0 A wavelength (room-temperature structure) using SHELXC/D/E

(Sheldrick, 2010; Table 1). The initial models for C domain were built in

COOT (Emsley et al., 2010).

The B-B protein solution was prepared at 20 mg/ml from lyophilized

protein and mixed in a 1:1 ratio with crystallization solution (60 mM MES

[pH 6], 3.0 M ammonium sulfate, and 2.5% [v/v] glycerol). Diffracting crystals

formed by hanging-drop vapor diffusion within two months and were of the

space group P65. Cryopreservation of the B-B crystal was achieved by trans-

ferring to sodium malonate (3.4 M and pH 6), and the crystal was frozen by

direct immersion in liquid nitrogen. All data were collected at the APS at

Argonne National Laboratory, beamline 22-ID (SER-CAT), at 1.0 A wave-

Structure 22, 1467–

length. Data were processed and scaled with HKL2000 (Otwinowski and

Minor, 1997; Table 1).

The B-B structurewas solved bymolecular replacement in PHASER (McCoy

et al., 2007) using the cryogenic C-domain structure as the search model,

which has a 91% sequence similarity to each domain of B-B. The search

model was modified to remove any alternative conformations and ten mobile

residues at the N terminus. The initial model for the individual domains of

B-B was built by AUTOBUILD (Terwilliger et al., 2008) and then rebuilt in

COOT (Emsley et al., 2010). The residues in the interdomain linkers were

manually built in COOT (Emsley et al., 2010).

Refinement and Heterogeneous Model Building

Refinement of all models was carried out in phenix.refine (Adams et al., 2010).

Initial refinement was performed using two isotropic B factors per residue.

Once themodel was placed, it was refined using isotropic Bs for all atoms (Fig-

ure S2A). After several rounds of iterative refinement and model building, the

visible concerted anisotropy was used to fit main chain alternative conforma-

tions in segments (e.g., helix1). Each alternative-conformation segment was

rotated and translated in COOT (Emsley et al., 2010) to fit the direction of

anisotropy in the carbonyl oxygen 2Fo-Fc density, effectively separating these

alternative conformations (Figure S2B). The side chain for each alternative

conformation was then fit down to 0.3–0.5s levels. Candidates that could not

be fit rotamerically or that later refined to zero occupancy were discarded. To

avoid bad geometry across peptide bonds, backbone alternative conforma-

tionswere continued to the flanking Ca atoms if needed, using backrubs (Davis

et al., 2006) in a newPHENIX (Adams et al., 2010) utility written for this purpose.

After all main chain and side chain alternative conformations were fit, several

rounds of occupancy and anistropic B-factor refinement were performed. All

atoms but H were refined as anisotropic for the cryogenic C-domain structure,

and all atoms but H and waters were refined as anisotropic for the room-

temperature and B-B structures. The 2Fo-Fc and Fo-Fc density at each residue

was checked after each round of refinement. Final refinement for all structures

was carried out in phenix.refine using automatic weight optimization. The final

refined models for the cryogenic structure and room-temperature structure

had Rwork/Rfree values of 11.0%/12.7% and 11.2%/14.1%, respectively,

whereas the final refined model for the B-B structure had Rwork/Rfree values

of 14.2%/18.5%.

Illustrations

Coordinate files for molecules in crystal contact were produced in Chimera

(Pettersen et al., 2004). Their all-atom contact dots were produced by the

interface feature in MolProbity (Chen et al., 2010), counted in Mage (Richard-

son and Richardson, 2001), and converted to square angstroms by dividing

by the default contact-dot density of 16/A2. Figure 5 was made in KiNG

(Chen et al., 2009). Figure 4A was constructed using Prekin and Mage. All

remaining structural illustrations were made in PyMOL (http://www.pymol.

org/). Figure 2C was made in Mathematica (2010).

ACCESSION NUMBERS

The Protein Data Bank accession numbers for the molecular coordinates

and structure factors reported in this work are 4NPD (cryogenic C domain),

4NPE (room temperature C domain), and 4NPF (B-B).

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

four figures, and one table and can be found with this article online at http://

dx.doi.org/10.1016/j.str.2014.08.014.

AUTHOR CONTRIBUTIONS

Y.Q. and A.H. performed all protein expression and purification. L.N.D. per-

formed all crystallization. L.N.D. and C.W.P. contributed to data collection,

structure solution, and refinement. L.N.D., C.W.P., J.S.R., and T.G.O. analyzed

and interpreted the structures and discussed the results. D.C.R. and J.S.R.

made and analyzed the helix-axis and noncognate superpositions, D.C.R.,

1477, October 7, 2014 ª2014 Elsevier Ltd All rights reserved 1475

Structure

Conformational Heterogeneity of Protein A

J.S.R., and L.N.D. analyzed crystal contacts; and L.N.D., C.W.P., D.C.R.,

J.S.R., and T.G.O. made figures. L.N.D. and T.G.O. wrote the initial manuscript

draft; and L.N.D., C.W.P., D.C.R., J.S.R., and T.G.O. revised it.

ACKNOWLEDGMENTS

This paper is dedicated to the memory of the late Prof. Thomas Alber, whose

vision led us to see proteins in a new and illuminating way. Crystal screening

and data processing were conducted in collaboration with the Duke Macro-

molecular X-ray Crystallography Shared Resource. Diffraction data were

collected remotely at the Southeast Regional Collaborative Access Team

22-ID beamlines at the Advanced Photon Source, Argonne National Labora-

tory, supported by the US Department of Energy Sciences under contract

number W-31-109-Eng-38. The plasmid construction, protein expression,

and purification aspects of this work were supported by NIH grant R01-

GM081666 (to T.G.O.). The crystallographic and structure analysis aspects

of this work were supported by NIH grant R01-GM073930 and GM073919

(to D.C.R.) and in part by NIH grant P01-GM063210 Project IV (to J.S.R.).

We thank Nigel Moriarty for the utility in PHENIX that spreads backbone alter-

native conformations to the flanking Ca atoms.

Received: June 24, 2014

Revised: August 12, 2014

Accepted: August 17, 2014

Published: October 7, 2014

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