A Disulfide-Free Single-Domain VL Intrabody with Blocking Activity towards Huntingtin Reveals a...

doi:10.1016/j.jmb.2011.09.034 J. Mol. Biol. (2011) 414, 337–355

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Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

A Disulfide-Free Single-Domain VL Intrabody withBlocking Activity towards Huntingtin Reveals aNovel Mode of Epitope Recognition

André Schiefner 1, Lorenz Chatwell 1, Jana Körner 2, Irmgard Neumaier 1,David W. Colby3, Rudolf Volkmer 2, K. Dane Wittrup3, 4 and Arne Skerra 1⁎1Munich Center for Integrated Protein Science and Lehrstuhl für Biologische Chemie, Technische UniversitätMünchen,85350 Freising-Weihenstephan, Germany2Institut für Medizinische Immunologie, Charité, 10115 Berlin, Germany3Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA4Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

Received 18 July 2011;received in revised form19 September 2011;accepted 20 September 2011Available online24 September 2011

Edited by I. Wilson

Keywords:Huntington's disease;immunoglobulin;poly-glutamine;protein aggregation;protein engineering

*Corresponding author. Lehrstuhl fü85350 Freising-Weihenstephan, GermPresent address: D. W. Colby, DepAbbreviations used: BSA, buried

ethylenediaminetetraacetic acid; HBimmobilized metal affinity chromatovariable fragment; SPR, surface plas

0022-2836/$ - see front matter © 2011 E

We present the crystal structure and biophysical characterization of ahuman VL [variable domain immunoglobulin (Ig) light chain] single-domain intrabody that binds to the huntingtin (Htt) protein and has beenengineered for antigen recognition in the absence of its intradomaindisulfide bond, otherwise conserved in the Ig fold. Analytical ultracentri-fugation demonstrated that the αHtt-VL 12.3 domain is a stable monomerunder physiological conditions even at concentrations N20 μM. Usingpeptide SPOT arrays, we identified the minimal binding epitope to beEKLMKAFESLKSFQ, comprising the N-terminal residues 5–18 of Htt andincluding the first residue of the poly-Gln stretch. X-ray structural analysisof αHtt-VL both as apo protein and in the presence of the epitope peptiderevealed several interesting insights: first, the role of mutations acquiredduring the combinatorial selection process of the αHtt-VL 12.3 domain—initially starting from a single-chain Fv fragment—that are responsible forits stability as an individually soluble Ig domain, also lacking the disulfidebridge, and second, a previously unknown mode of antigen recognition,revealing a novel paratope. The Htt epitope peptide adopts a purely α-helical structure in the complex with αHtt-VL and is bound at the base of thecomplementarity-determining regions (CDRs) at the concave β-sheet thatnormally gives rise to the interface between the VL domain and its pairedVH (variable domain Ig heavy chain) domain, while only few interactionswith CDR-L1 and CDR-L3 are formed. Notably, this noncanonical mode ofantigen binding may occur more widely in the area of in vitro selectedantibody fragments, including other Ig-like scaffolds, possibly even if a VHdomain is present.

© 2011 Elsevier Ltd. All rights reserved.

r Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5,any. E-mail address: [email protected] of Chemical Engineering, University of Delaware, Newark, DE 19716, USA.surface area; CDR, complementarity-determining region; EDTA,S, Hepes-buffered saline; HD, Huntington's disease; Htt, huntingtin; IMAC,graphy; PDB, Protein Data Bank; PEG, polyethylene glycol; scFv, single-chainmon resonance; TBS, Tris-buffered saline.

lsevier Ltd. All rights reserved.

mailto:[email protected]

http://dx.doi.org/10.1016/j.jmb.2011.09.034

338 Crystal Structure of an αHtt-VL Domain

Introduction

Huntington's disease (HD) is a severe autosomaldominant neurodegenerative disorder affecting 4–10in 100,000 individuals in the Western world. 1

Neuropathology of HD displays a loss of neuronsin the caudate and putamen (striatum), along withthe deposition of aggregated huntingtin (Htt) pro-tein. The Htt gene, encoding a 347-kDa cytoplasmicelongated HEAT (Huntingtin, elongation factor 3,protein phosphatase 2A, and the yeast PI3-kinaseTOR1) repeat protein,2 is widely expressed andnecessary for normal development.3HD is caused by an expanded, unstable CAG

trinucleotide repeat in the Htt disease gene, whichtranslates as a poly-glutamine repeat with variablelength in the encoded protein. Onset and clinicalcourse depend on the degree of poly-Gln repeatexpansion, with longer expansions resulting inearlier onset and more severe clinical manifestation.An unusual polymorphism in the number oftrinucleotide repeats has been identified in thenormal human population, whereby repeat numbersin excess of 40 have been described as pathological.The adjacent poly-Pro region is also polymorphicand varies from 7 to 12 residues;4 however, itsimplication in HD pathology has not been proven.Intracellular Htt aggregates were reported in post

mortem brain samples of HD patients,5 consistingmainly of the amino-terminal part of Htt andoccurring in both the cytoplasm and the nucleus ofneurons. The expanded poly-Q region, which isusually associated with the observed Htt aggre-gation6 and seems to impair intracellular proteindegradation,7 is clearly involved in the diseasemechanism.8 In particular, anN-terminal proteolyticfragment of Htt is prone to aggregation and exertscytotoxic activity.9 Furthermore, the probability ofaggregate formation is a strong function of poly-Qlength.10

Although the physiological role of Htt and itsexact mechanism of neuronal pathogenicity are stilla matter of ongoing investigation,9 it is wellestablished that antibodies directed against Htt canpartially ameliorate aggregation and toxicity. Thiswas demonstrated in a cell-free aggregation assay11

and in several cellular HD models.12,13 Consequent-ly, smaller cognate binding proteins may have asimilar inhibitory effect while circumventing thedisadvantages encountered by the use of full-sizeantibodies—or larger immunoglobulin (Ig) frag-ments—especially with respect to biosynthesis inthe reducing intracellular environment, which ham-pers disulfide bond formation, and potential immu-nogenic side effects.To this end, an intrabody variable light chain with

binding specificity towards the N-terminal MATLE-KLMKAFESLKSFQQQ sequence of Htt was engi-neered. This VL (variable domain Ig light chain)

domain was originally isolated via yeast cell surfacedisplay from a nonimmune human single-chainvariable fragment (scFv) antibody library,14,15 fol-lowed by in vitro affinity maturation. During theinitial selection experiments, it was found that theparatope was mainly localized in the λ light chain ofthe scFv fragment, and one clone that was seren-dipitously isolated and found to encode just the Vλ2.4.3 domain showed improved intracellular expres-sion with comparable target affinity.To mimic the reducing intracellular environment

of the cytoplasm, where Htt is normally expressed,the conserved disulfide bond in this Ig V-domainwas subsequently eliminated via the amino acidreplacements CL23V and CL88A.16 However, this ledto a drastic loss in affinity (KDN10 μM), whilefunctional yeast cell surface expression and detect-able inhibitory activity on intracellular Htt aggrega-tion were retained. After three cycles of randommutagenesis and selection for improved binding tothe Htt epitope peptide, the αHtt-VL domain 12.3was obtained, which carries the additional muta-tions FL36I, YL50D, KL66R, and AL74T and hasapproximately 3 nM affinity as estimated fromFACS titration.17

This engineered αHtt-VL intrabody was shown torescue toxicity in a neuronal model of HD and toinhibit Htt aggregation and toxicity in a Saccharo-myces cerevisiae HD model.17 It appears to besignificantly more potent than earlier anti-Httintrabodies and, thus, provides a potential candi-date for gene therapy treatment of HD. To provide abasis for further improvement of this Htt aggrega-tion inhibitor and to gain insight into its molecularmechanism, we present a biophysical and structuralanalysis of the αHtt-VL domain both in its uncom-plexed state and in association with the cognate Htttarget peptide.

Results

Intracellular expression of the αHtt-VL domain inEscherichia coli as a highly soluble protein andits biophysical characterization

The αHtt-VL 12.3 domain was produced in thecytoplasm of E. coli as a fusion protein both with a C-terminal His6-tag

18 (αHtt-VL-his) andwith the Strep-tag19 (αHtt-VL-strep). The recombinant protein waspurified to homogeneity from the soluble whole cellextract either by immobilized metal affinity chro-matography (IMAC) or by streptavidin affinitychromatography, followed by gel filtration (Fig. 1).While the engineered VL domain was recovered as asoluble and apparently monomeric protein usingeach of the two affinity tags, the purified proteinshowed some tendency to aggregate and adsorb to

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45.0 –

35.0 –

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Fig. 1. Purity and oligomeric state of the bacteriallyproduced αHtt-VL. (a) SDS-PAGE (15%) illustrating thepurification of αHtt-VL-his. Lane 1, soluble whole cellextract of E. coliK-12 JM83 harboring pASK75-cVLhis after8 h of induction of gene expression; lane 2, protein elutedfrom IMAC on a Zn(II)-charged IDA-Sepharose column ina gradient at elevated imidazole concentration; lane 3,purified protein after gel filtration on a Superdex 75column in the presence of 10 mM Tris–HCl (pH 8.0). (b)Analytical ultracentrifugation of αHtt-VL-his. The sample[23 μM protein solution in 10 mM Tris–HCl (pH 8.0)] wascentrifuged at 4 °C and 20,000 rpm in a Beckman OptimaXL-I analytical ultracentrifuge with Ti-60 rotor until theconcentration gradient, which was monitored via UVabsorption at 280 nm, reached equilibrium. Curve fit of thedata using a one-species model (see Materials andMethods) led to a calculated mass of 15.7±0.4 kDa.

339Crystal Structure of an αHtt-VL Domain

chromatography columns in the presence of highersalt concentration (≥150 mM NaCl). Interestingly,its elution behavior during size-exclusion chroma-tography in a low salt buffer was slightly erratic andtypically yielded two peaks, one with an apparent

size roughly as large as expected and one withstronger retardation.Hence, to gain further insight into the oligomer-

ization state of αHtt-VL in solution, we performedanalytical ultracentrifugation of αHtt-VL-his in thepresence of 10 mM Tris–HCl (pH 8.0) up to a proteinconcentration of 23 μM (Fig. 1b). Under theseconditions, the engineered VL domain showed auniform concentration profile, which was well fittedunder the assumption of a monomeric species with15.7±0.4 kDa, that is, a value close to its calculatedmass of 12.8 kDa. Thus, despite the high proteinconcentration in this experiment, there was no signof dimer formation, which is a phenomenonotherwise often observed for Ig light chain frag-ments of the Bence-Jones type.20

Functional binding assay, epitope analysis,and affinity measurement for αHtt-VL

Binding activity of αHtt-VL was first tested in anELISA with the C-terminally biotin-labeled epitopepeptide MATLEKLMKAFESLKSFQQQK, corre-sponding to the N-terminal region of human Htt(Fig. 2). The same sequence had been used for the invitro selection of the engineered VL domain.15 Whenthe purified αHtt-VL-strep was preadsorbed to amicrotiter plate and incubated with the biotinylatedHtt peptide, followed by an avidin-enzyme conju-gate, a pronounced saturation curve was detected,which was fitted with an apparent dissociationconstant (KD) of 182 nM.As this 21-residue peptide was considerably

longer than one would expect for a typical linearepitope,21 we performed a substitution and dele-tion analysis using arrays of synthetic peptideswith systematically variegated amino acid se-quences, which were coupled to a cellulosemembrane according to the SPOT method22 (Fig.2b). These membranes were probed with αHtt-VL-strep, whose affinity tag permitted direct detectionvia a streptavidin-enzyme conjugate.19 The substi-tution analysis indicated a specific pattern for thesequence region EKLMKAFESLKSFQQQ, whereassystematic truncation of the full-length peptidefrom both ends resulted in the minimal sequenceEKLMKAFESLKSFQ (Fig. 2c). Thus, it becameclear that, apart from the N-terminal region ofHtt, only the first amino acid of its poly-Q stretch(starting with residue number 18; cf. NCBI acces-sion code NP_002102) was essential for recognitionby αHtt-VL.To further confirm and narrow down the central

epitope, we synthesized a group of soluble peptidesencompassing the minimal sequence LMKA-FESLKSFQ, together with more extended sequencesup to the one identified in the substitution analysis,and we investigated their binding to the immobi-lized αHtt-VL-strep by real-time surface plasmon

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- A D E F G H I K L M N P Q R S T V W YMATLEKLMKAFESLKSFQQQK

TLEKLMKAFESLKS

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ATLEKLMKAFESLK

MATLEKLMKAFESLKSFQQQK

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ATLEKLMKAFESLKSFQQQ

TLEKLMKAFESLKSFQQQK

MATLEKLMKAFESLKSFQ

ATLEKLMKAFESLKSFQQ

TLEKLMKAFESLKSFQQQ

LEKLMKAFESLKSFQQQK

MATLEKLMKAFESLKSF

ATLEKLMKAFESLKSFQ

TLEKLMKAFESLKSFQQ

LEKLMKAFESLKSFQQQ

EKLMKAFESLKSFQQQK

(a)

(c)

(b)

Fig. 2. Htt peptide binding studies and SPOT epitope analysis. (a) ELISA for peptide-binding activity of αHtt-VL-strep. Amicrotiter platewas coatedwith thepurified recombinant protein,washed, and incubatedwith adilution series of thepeptideMATLEKLMKAFESLKSFQQQK-(Nɛ)–biotin, followed by detection with ExtrAvidin-AP (alkaline phosphatase) conjugate.αHtt-VL-strep shows saturation binding behavior for the peptide (circles), with an apparentKD value of 182±35 nM,whereasonlyweakbackground signalsweredetected in the absence of theVLdomain (squares). (b) Epitope substitution analysiswithan array of variant MATLEKLMKAFESLKSFQQQK peptides synthesized on cellulose (with the C-terminus covalentlyimmobilized via a hydrophilic spacer): each residue (left margin, from top to bottom) was systematically substituted by allamino acids, except for Cys (- indicates the original amino acid). The membrane was incubated with purified αHtt-VL-strepand washed; bound protein was detected with StrepTactin-HRP conjugate. The pattern indicates that the four N-terminalpositions and the ultimate C-terminal residue, as well as Phe at position number 11, tolerate most side chains. (c) SPOTepitope truncation analysis using the same setup as in (b). Binding signals clearly fade as soon as the immobilized peptidefrom either side becomes shorter than the minimal epitope sequence EKLMKAFESLKSFQ.


resonance (SPR) measurements. The dissociationconstants resulting from the corresponding equilib-rium binding analyses revealed the lowest values,between 150 and 250 nM, for the peptidesEKLMKAFESLKSFQ1–3 (i.e., versions carrying oneto three C-terminal Gln residues). In contrast,complete deletion of the Gln residues, or shortening

of the peptide by one or two amino acids from theN-terminal side, led to a drastic increase in the KDvalue up to the micromolar range.Thus, the sequence EKLMKAFESLKSFQ (residues

5–18 of native Htt), with a central recognition motifAxESLxxF appearing from the substitution analysis(cf. Fig. 2b), constitutes the minimal epitope recog-


nized by αHtt-VL. Consequently, this peptide waschosen for subsequent co-crystallization experi-ments (see below). On the other hand, as thepeptide EKLMKAFESLKSFQQQ had similar affin-ity and was the one with the longest stretch of Glnresidues from the oligomeric repeat of Htt investi-gated here, this version was chosen for a moredetailed SPR analysis (Fig. 3). To enhance sensitiv-ity, we inverted the setup compared with the earliermeasurements: this time, the peptide was covalent-ly attached to the sensor chip via an N-terminal Cysresidue (for details, see Materials and Methods)while αHtt-VL-strep was applied in the fluid phase(Fig. 3a). The kinetic analysis resulted in parameterskon=3.86×10

4 M−1 s−1 and koff=1.73×10− 3 s−1 ,

leading to a corresponding KD value of 45 nM. Thisvalue was in close agreement with the dissociation

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Concentration αHtt-VL [nM]

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Fig. 3. SPR analysis of epitope peptide binding by αHtt-VL. (a) Real-time BIACORE-X measurements with a CM5sensor chip carrying the covalently attached C-Ahx-EKLMKAFESLKSFQQQ peptide while the purifiedαHtt-VL-strep protein was applied at varying concentra-tions between 250 nM and 4 nM (in a 1:2 dilution series).(b) Equilibrium analysis with a plot of the plateauresonance unit values (at about 1700–1800 s) from (a)against the applied αHtt-VL concentration. Hyperboliccurve fit resulted in a KD value of 44.4±1.1 nM.

constant derived from an alternative equilibriumanalysis of the same data: KD=44.4±1.1 nM.

Crystallization and structure determination ofαHtt-VL and αHtt-VL/Htt5–18

The uncomplexed (apo) αHtt-VL domain [MGS-VL(1–111)-AH6] was crystallized in the presence of(NH4)2SO4 at pH 9.8 (for details, see Materials andMethods). The crystals diffracted to a resolution of2.05 Å and belonged to the space group P41212,containing two αHtt-VL monomers per asymmetricunit (Table 1). Interpretable electron density wasobserved for residues −1 to 113 in monomer A andfor residues 3–111 in monomer B. Both monomersshowed a very low mutual root-mean-square devi-ation (RMSD) of 0.24 Å upon pairwise superpositionof 109 Cα atoms (residues 2–110 of αHtt-VL).The complex of αHtt-VL-his with the EKLMKA-

FESLKSFQ epitope peptide (Htt5–18) was crystal-lized at a protein:peptide molar ratio of 1:4 in thepresence of polyethylene glycol (PEG) 600 atpH 4.1. Crystals diffracting to a resolution of2.6 Å belonged to the space group P21 andcontained eight αHtt-VL/Htt5–18 complexes in theasymmetric unit. Interpretable electron density wasobserved at least for residues 2–110 in all αHtt-VLmonomers and always for the entire bound Htt5–18epitope peptide. A few additional residues weretraced for chains E, I, O, and M (with chain Erepresenting the most complete model, comprisingresidues −1 to 111). The eight αHtt-VL/Htt5–18complexes showed almost identical structures, witha mutual RMSD of less than 0.49 Å over 109 Cα

positions. The B-values of the bound Htt5–18 peptidewere slightly increased, on average, by 14.4 Å2, incomparison with αHtt-VL (Table 1).The most well defined polypeptide chains of each

crystal structure, that is, monomerA forαHtt-VL andcomplex AB for αHtt-VL/Htt5–18, were chosen forfurther analysis and structural interpretation. Themutual RMSD of αHtt-VL between apo and holostructures was 0.59 Å (again, upon pairwise super-position of the 109 Cα atoms), indicating highconformational similarity and, in particular, theabsence of an induced fit for the three hypervariableloops (complementarity-determining regions,CDRs), which all showed well-defined electrondensity and mostly identical side-chain rotamers.The low RMSD value for the superposition of apoαHtt-VL with αHtt-VL/Htt5–18 is in the range ofRMSD values observed among the eight αHtt-VL/Htt5–18 complexes within the asymmetric unit of theP21 crystal structure. Only residues SerL7 and ProL8

show a displacement larger than 2 Å for their Cα

positions. These residues are involved in differentcrystal packing environments but are irrelevant forepitope recognition.

Table 1. X-ray data collection and refinement statistics

αHtt-VL αHtt-VL/Htt5–18

Data collectionSpace group P41212 P21Unit cell parameters

a, b, c (Å) 107.68, 107.68,50.62

53.09, 89.66,95.00

α, β, γ (°) 90, 90, 90 90, 99.45, 90Number of protein/peptide

molecules per asymmetricunit

2/0 8/8

Wavelength (Å) 1.5418 1.5418Resolution (Å) 30–2.05

(2.10–2.05)a30–2.60

(2.70–2.60)a

Completeness (%) 99.8 (100) 88.7 (92.0)Unique reflections 19,201 (1301) 24,090 (2651)Multiplicity 15.0 (14.3) 2.8 (2.7)Mean I/σ(I) 32.7 (11.7) 9.8 (4.1)Rmeas (%)b 7.5 (27.3) 13.8 (38.7)Wilson B-factor (Å2) 23.5 18.8

RefinementResolution (Å) 20.0–2.05

(2.10–2.05)a19.86–2.60(2.67–2.60)a

Reflections (working) 18,215 (1304) 22,868 (1720)Reflections (test) 986 (69) 1221 (101)Rcryst (%)c 17.1 (17.2) 21.6 (24.8)Rfree (%)d 19.7 (24.5) 27.8 (33.3)Number of atoms protein/

ligands/waters1649/13/155 6504/944/204

B-values of protein/ligands/waters (Å2)

17.7/40.4/20.2 22.6/37.0/10.0

Ramachandran plot:favored/outliers (%)e

96.4/0.0 96.7/0.0

RMSD bonds (Å)/angles (°) 0.015/1.37 0.005/0.83a Values in parentheses represent the numbers for the highest-

resolution shell.b Rmeas =

Phkl

ffiffiffiffiffiffiffin

n − 1

p Pn

j = 1j Ihkl;j − hIhkli jP

hkl

PjIhkl;j

.

c Rcryst =P

hkljFobshkl − Fcalchkl jP

hklFobshkl

.d Rfree is calculated as for Rcryst but using 5% of the reflections

excluded from refinement.e The Ramachandran plot was calculated with MolProbity.23

Calculation with PROCHECK24 resulted in a typical outlier in theCDR-L2, which is frequently observed in other Ig structures, too.


The canonical structures of CDR-L1, CDR-L2, andCDR-L3 were assigned based on the amino acidsequences as 5, 1, and 5, respectively.25,26 Furthercomparison with the crystal structure of the Fabfragment of the myeloma protein KOL [Protein Data

Fig. 4. Structure of the αHtt-VL/Htt5–18 complex. (a) Stereoloops CDR-L1, CDR-L2, and CDR-L3 highlighted in green,EKLMKAFESLKSFQ (Htt5–18; salmon) is bound in the groove finvolved in the association with the VH domain in intact antibare formed by the N-terminal part of Htt5–18, whereas its C-terminal Gln residue (GlnP18), that is, the first residue of the pchains of αHtt-VL engaged in contacts with Htt5–18 and the firselectron density map of Htt5–18 was contoured at 1 σ and is shodefinition. (c) Superposition between αHtt-VL/Htt5–18 and apothe CDRs are depicted as sticks, and β-strands are labeled acframework (FW) regions as well as residues of the CDRs thatαHtt-VL are labeled.

Bank (PDB) entry 2FB4],27 which is a representativeof this λ light chain class, showed that the three CDRloops of αHtt-VL adopt the same canonical back-bone conformations, even with mostly identicalside-chain rotamers.

Structure of αHtt-VL in the apo form

The tertiary structure of αHtt-VL reveals the typicalfold of a variable Ig domain, comprising a sandwichof two antiparallel β-sheets (Fig. 4). Generally, thereare two different β-strand compositions found for V-type Ig domains: 4+5 and 3+6. These are distin-guished by the participation of strand a in either theβ-sheet deb or the β-sheet gfcc′c″.28 In the apo αHtt-VL crystal structure, β-strand a partly (residues 5and 6) participates in sheet deb, but it also associateswith sheet gfcc′c″, separated by a bulge (residues9–11; Fig. 4c).Themajor difference between theαHtt-VL structure

and the standard V-domain Ig fold is the absence ofthe canonical disulfide bond connecting β-strandsb and f from both sheets at the center of theβ-sandwich. In αHtt-VL, the corresponding Cysresidues L23 and L88 had been replaced by Val andAla, respectively.17 Although, normally, this disulfidebond is known to be crucial for the stabilization of theIg fold,29 its substitution (combined with the muta-tions that restored binding affinity; see Discussion)did not lead to relevant changes in the overalltopology (Fig. 5) or prevent stable folding of αHtt-VL,both on the yeast surface and in the yeast17 orbacterial cytoplasm (as demonstrated here).A DALI31 search for structural homologues

revealed the light chain of the αHIV-1 gp120-reactive Ig Fab fragment E51 (PDB entry 1RZF)30

as the most similar structure, with a Z-score of22.3, having 71 identical residues and an RMSDof 0.80 Å over the 109 Cα positions in commonwith αHtt-VL.

Structure of the αHtt-VL/Htt5–18 complex

In the crystal structure of αHtt-VL/Htt5–18, thecomplex between chains A and B exhibits thelargest buried surface areas (BSAs) of 551 Å2 and627 Å2, respectively, for αHtt-VL and Htt5–18,

representation of αHtt-VL in gray with the hypervariablecyan, and yellow, respectively. The Htt epitope peptideormed by the β-sheet gfcc′c″ of αHtt-VL, which is normallyodies. Most of the contacts between Htt5–18 and the CDRsterminal part interacts with framework residues. The C-oly-Q stretch of Htt, is most distant from the CDRs. Sidet and last residue of the peptide are labeled. (b) The 2Fo−Fcwn as a blue mesh around the peptide to illustrate its highαHtt-VL (gray and light blue, respectively). Side chains of

cording to the nomenclature for Ig V-domains. CDRs andshow detectable differences between αHtt-VL/Htt5–18 and


compared with the corresponding average BSAvalues of 531 Å2 and 598 Å2 for all eight complexesin the asymmetric unit. In complex with αHtt-VL,the Htt5–18 peptide adopts an entirely α-helicalsecondary structure, in line with previous structuralanalysis.32 Unexpectedly, it is bound at the surface

(a)

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N

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GlnP18

GluP5

N

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GlnP18

GluP5

ThrL32

TrpL96

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TyrL49

PheL98

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TyrL87

GluL45ProL44

Fig. 4 (legend on

of the β-sheet gfcc′c″, which constitutes an unusualparatope. While antigen binding is typicallymediated by the hypervariable loops (CDRs) atthe wedge-shaped tip of the Ig domain, αHtt-VLaccommodates this epitope peptide in a grooveapproximately at the focus of the concave β-sheet,

N

C

a

b c def

g

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DR-L1

CDR-L2

CDR-L3

TrpL91

GluP5

GlnP18

ProL44 GluL45TyrL87

IleL36 LeuL46

PheL98

TyrL49AsnL34

AspL50TrpL96

ThrL32

AspL50

AsnL34

TrpL96

previous page)

AlaL88

C

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ProL56

ArgL42

GluL45

AlaL55

ValL23

IleL36

αHtt-VL

ValL23

CysL23

CysL88

E51 VL

AlaL88

ProL2

SerL7

AlaL13

MetL48

ThrL32

ThrL90

SerL30AsnL27

ArgL66

AspL50

ThrL74

Fig. 5. Unique structural features of αHtt-VL compared to conventional Ig VL domains. (a) The canonical centraldisulfide bond in αHtt-VL has been eliminated by protein engineering: detailed view of the two substituted side chainsValL23 and AlaL88 with a corresponding 2Fo−Fc electron density map, contoured at 1 σ. For comparison, the same regionin the VL domain of the Fab fragment E51 (PDB entry 1RZF)30 is shown below, revealing the natural disulfide bridge. Thebackbone structure around the substituted Cys residues is essentially unchanged in αHtt-VL, indicating absence ofstructural rearrangement. (b) All residues that were mutated during the engineering of αHtt-VL and differ from thehuman germline V1–11 are highlighted as sticks. Residues introduced during initial yeast display selection are coloredblue (SL2P, PL7S, EL13A, SL27N, NL30S, AL32T, KL42R, KL45E, IL48M, PL55A, SL56P, and AL90T), the substituted residues ofthe disulfide bridge are highlighted in magenta (CL23V and CL88A), and residues that were introduced during affinitymaturation of αHtt-VL are depicted in orange (FL36I, YL50D, KL66R, and AL74T). Note that the central position L36 wasreplaced once during initial yeast display selection (YL36F) and then again during affinity maturation.15,17


in the direction from β-strands c″ to g (Fig. 4a).Notably, the same β-sheet surface is usuallyinvolved in the tight interface between the pairedVL and VH (variable domain Ig heavy chain)domains of an intact antibody.The Htt5–18 peptide forms a total of 193 contacts

(within 4.5 Å) to αHtt-VL, comprising 3 hydrogenbonds and 190 van der Waals contacts (Table 2). Astriking feature of this mostly hydrophobic andstructurally unconventional mode of epitope recog-nition is that the framework residues of αHtt-VLcontribute 64% of all contacts, while only 36%involve the hypervariable loops, in particular,CDR-L2. The three hydrogen bonds are mediated,respectively, via the CDR-L1 residues ThrL32 andAsnL34 to peptide side chains LysP6 and SerP13 andvia the CDR-L3 residue TrpL96 to GluP12. The contactinterface is mainly hydrophobic and dominated bythe peptide residues LeuP7, AlaP10, PheP11, LeuP14,and PheP17. Additional van der Waals main-chainand side-chain contacts are mediated through LysP6,

LysP9, SerP13, and SerP16. GluP12 forms only main-chain contacts with TrpL96, and GlnP18 forms onlyside-chain contacts with AlaL43 and ProL44. Peptideresidues GluP5, MetP8, and LysP15 do not form anycontacts to αHtt-VL.Two hydrophobic patches are prominent on αHtt-

VL: one comprising residues LeuL46, TyrL49, andProL56 and one arising from residues IleL36, ProL44,TyrL87, and PheL98. These two patches interact withthe residue clusters LeuP7, AlaP10, and PheP11 andLeuP14, PheP17, and GlnP18, respectively, of Htt5–18.PheP17 appears to be the most intimately boundresidue of the epitope peptide, contributing 23% ofall contacts and 19% of the BSA. On the other hand,TrpL96 located at the C-terminal end of CDR-L3contributes most of the contacts by αHtt-VL. A criticalrole of TrpL96 has been noted already during theiterative selection of αHtt-VL.

15 Interestingly, residuesPheP17 and TrpL96 do not contact each other, but bothare essential for complex formation as they togethergive rise to 43% of all observed contacts (cf. Table 2).

Table 2. Interactions between the VL domain and the bound antigen peptide in the αHtt-VL/Htt5–18 complex

Htt5–18 αHtt-VL

Residue BSA (Å2)

Contacts to αHtt-VL≤4.5 Å

Residue BSA (Å2)

Contacts to Htt5–18≤4.5 Å

Number Residues in αHtt-VL Number Residues in Htt5–18

GluP5 — — — AsnL31 (L1) 3.80 — —⁎LysP6 79.02 21 ⁎ThrL32, TyrL49, AspL50 ⁎ThrL32 (L1) 27.33 6 ⁎LysP6

LeuP7 74.30 24 LeuL46, TyrL49, ProL56 ⁎AsnL34 (L1) 25.87 12 AlaP10, ⁎SerP13

MetP8 — — — IleL36 42.76 11 LeuP14, PheP17

LysP9 54.83 11 TrpL96 GlnL38 2.95 — —AlaP10 53.91 16 AsnL34, LeuL46, TyrL49 AlaL43 5.36 1 GlnP18

PheP11 31.73 7 LeuL46 ProL44 42.71 18 LeuP14, PheP17, GlnP18⁎GluP12 10.63 4 ⁎TrpL96 GluL45 7.98 6 LeuP14

⁎SerP13 67.83 24 ⁎AsnL34, TrpL96, PheL98 LeuL46 73.66 18 LeuP7, AlaP10, PheP11, LeuP14

LeuP14 73.01 19 IleL36, ProL44, GluL45, LeuL46 TyrL49 55.20 26 LysP6, LeuP7, AlaP10

LysP15 — — — AspL50 (L2) 26.25 10 LysP6

SerP16 33.90 13 TrpL96, PheL98 LeuL53 (L2) 0.67 — —PheP17 118.18 44 IleL36, ProL44, TyrL87, PheL98 AlaL55 (L2) 0.84 — —GlnP18 30.63 10 AlaL43, ProL44 ProL56 (L2) 34.82 3 LeuP7

TyrL87 21.23 13 PheP17

AlaL89 (L3) 3.51 — —TrpL91 (L3) 17.91 — —⁎TrpL96 (L3) 95.53 39 LysP9, ⁎GluP12, SerP13, SerP16

PheL98 60.66 30 SerP13, SerP16, PheP17

GlyL99 0.61 — —628.0(35.3% of theaccessible surfacearea, ASA)

193 549.6(9.4% ASA)

193

Residues labeled with asterisks are involved in hydrogen bonds. All values were determined by PISA.


The molecular surface of αHtt-VL is well adaptedto binding the Htt5–18 peptide epitope, as theirmolecular shapes—cylindrical for the α-helicalpeptide and concave for the exposed β-sheet surfaceof the VL domain—are almost perfectly comple-mentary, which results in a complexation signifi-cance score of 1.0 as calculated with PISA (ProteinInterfaces, Surfaces and Assemblies).33 As a conse-quence, 35% of the solvent-accessible surface ofHtt5–18 is buried in its complex with αHtt-VL.In contrast, the equivalent surface of the isolated

VL domain of the structurally related E51 Fabfragment, for example, does not exhibit a compara-ble groove to accommodate such a helical peptide.Instead, the E51 VL domain displays a rather flathydrophobic surface, which forms the interface withthe paired VH domain (Fig. 6). Three side chains areof special importance in creating the pronouncedgroove in αHtt-VL: Ile

L36 deepens a depression as itis smaller than the typical Tyr residue at thisposition, TrpL96 raises one edge of the groove, andthe nearby PheL98 side chain extends its base.Calculation of the surface electrostatics using

APBS (Adaptive Poisson–Boltzmann Solver) 35revealed that the interface between αHtt-VL andHtt5–18 is almost charge neutral, except for a slightlynegative potential in the area of CDR-L2 caused byAspL50. Compared with the VL domain of the Fabfragment E51, αHtt-VL appears to be generally moreacidic, in agreement with its calculated pI of 4.6. The

overall hydrophobicity of αHtt-VL seems to beslightly decreased in comparison with the E51 VLdomain, with hydrophobic side chains concentratedat three patches: (i) LeuL46, TyrL49, and ProL56; (ii)IleL36, ProL44, TyrL87, and PheL98; and (iii) TrpL96

and TrpL91. On the other hand, hydrophobic surfaceresidues are more evenly distributed on the E51 VLdomain (Fig. 6).

Oligomerization of the Ig domain in the αHtt-VLand αHtt-VL/Htt5–18 crystal structures

In line with its biophysical characterization, boththe free and the complexed forms of αHtt-VLapparently crystallize as monomeric proteins asjudged from PISA analysis, resulting in maximalcomplexation significance score values of 0.0 and0.02, respectively, for interdomain contacts. How-ever, in both crystal structures, a peculiar mode ofcrystal packing was observed for the engineered VLdomain that showed certain resemblance withpreviously studied Ig domain dimers, yet differingfrom the typical homo-association in Bence-Jonesproteins such as for the myeloma protein RHE (PDBentry 2RHE)36 (Fig. 7). Contrasting with theconventional VL/VL association, which is structur-ally homologous to the VL/VH hetero-dimerizationin intact antibodies (or corresponding Fab and Fvfragments),37 αHtt-VL shows in the crystal a back-to-back/head-to-tail mode of association, with the

ValL96

AspL32

GluL50

SerL34

TyrL36

LysL45

TrpL96

ThrL32

AspL50

TyrL49

LeuL46

IleL36

AsnL34

TyrL87

PheL98

ProL44

GluL45

GlnP18

GluP5

αHtt-VL E51 VL

PheL98

GlnP18

TyrL87

ProL44

GluL45

TrpL96

ThrL32

GluP5

AspL50

TyrL49

AsnL34

LeuL46

IleL36

LysL45

TyrL36

SerL34

GluL50

AspL32

ValL96

(a) (b)

Fig. 6. Biophysical characteristics of the αHtt-VL paratope. (a) Electrostatic surface representation [from −10 kBT/e(red) to +10 kBT/e (blue)] for the Ig domain in the αHtt-VL/Htt5–18 complex compared with that of a conventional VLdomain such as from the Fab fragment E51 (PDB entry 1RZF). (b) Hydrophobic representation of the same surface regions.Residues are colored according to increasing hydrophobicity from green (hydrophilic) over gray (neutral, including thepolypeptide backbone) to brown.34 Residues of αHtt-VL forming five or more van der Waals contacts with Htt5–18(salmon) are labeled, as well as those residues of the E51 VL domain that are different.


CDRs of both monomers pointing into oppositedirections. Hence, the dyad symmetry axis linkingthe paired domains runs perpendicular to their β-sandwiches, whereas in the conventional mode ofVL dimerization, it is oriented essentially parallelwith the β-strands.Interestingly, the detailed mode of αHtt-VL/αHtt-

VL association differs for the two crystal structures

(Fig. 7). This indicates that the corresponding dimersare only stabilized by weak interactions, possiblydue to crystal packing, but should not be stable insolution. Superposition of the two different crystal-lographic αHtt-VL dimers requires a rotation of 56°and a translation of 14 Å to superimpose the secondmonomer. The average BSA on each monomer forthe putative dimers of αHtt-VL (634 Å

2) and of αHtt-

designed VL LENQ89LαHtt-VL/Htt5-18

αIGF-II antibody Fv αHEL4 human VH

αHtt-VL

Bence-Jones protein RHE

Fig. 7. Ig V-domain packing modes observed in protein crystals. Solid-state VL domain assemblies as observed forαHtt-VL and αHtt-VL/Htt5–18 are compared with other typical variable domain interactions from previous studies: theBence-Jones proteins RHE (2RHE) and LENQ89L (1QAC), a designed VL domain (PDB entry 1IVL), an αHEL4 human VHdomain (1OHQ), and an αIGF-II antibody (3KR3). To visualize the differing orientations, we show the β-sheet deba of eachβ-sandwich in darker gray. CDR loops of VL domains are colored bright green, cyan, and yellow, while the CDR loops ofVH domains are colored in corresponding darker shades.


VL/Htt5–18 (536 Å2) is considerably smaller than that

of the proto typical Bence-Jones dimer RHE (931 Å2)or of the VL/VH interface in the Fab fragment E51(718 Å2). Also, contrasting with the conventionalhydrophobic interface between the paired variabledomains of an antibody, this αHtt-VL/αHtt-VLassociation involves the predominantly hydrophilicsurface on the opposite side of each Ig domain.Notably, crystal structures of other engineered VLdomains,38 including the Q89L variant of the myelo-ma protein LEN,39 revealed a hydrophobic mode ofIg domain pairing involving essentially the sameinterface as for RHE but with an upside downmutualorientation, which is clearly different (cf. Fig. 7).

Discussion

Antibody fragments expressible in the cellularcytoplasm, so-called intrabodies, have been devel-oped against a host of medically relevant intracel-lular targets. In contrast to large Y-shapedantibodies with their four polypeptide chains,intrabodies usually are truncated versions compris-ing only the variable domains that are required forantigen recognition. These can be scFv fragments oreven isolated variable domains, both formats con-sisting of a single polypeptide chain that can beeasily expressed inside cells.40–43 However, it is wellknown that the canonical disulfide bond common toall Ig domains cannot readily form in the reducing

milieu within a cell,29 a phenomenon that consider-ably affects the stability of Ig domains. Nevertheless,depending on the overall thermodynamic stabilityof an scFv fragment, for example, the replacement ofthe conserved Cys residues can in principle betolerated, leading to a folded antibody fragmentwith antigen-binding activity.16,44

Beyond the scFv format, the construction of so-called single-domain antibodies has promptedincreasing interest during recent years, in particular,for potential applications as small monomericbinding proteins in medical therapy.45 Heavychain antibodies from cameloids, for example,allow the preparation of isolated VHH domainswith pronounced antigen-binding activities as novelreagents,46 meanwhile dubbed Nanobodies.47 SuchVHH antibodies lack the hydrophobic interfaceregion with the light chain as a result of a fewside-chain replacements, sometimes assisted byshielding via an extended CDR-H3 loop. Althoughit seems that Nanobodies can at least in part befunctionally expressed in the reducing cellularcytoplasm,48 an engineered intrabody that intrinsi-cally lacks disulfide bonds, such as the αHtt-VLprotein investigated here, should provide a clearadvantage.The biophysical analysis of the isolated recombi-

nant αHtt-VL domain in this study confirms its tightand specific binding activity for the Htt epitopepeptide, which was previously demonstrated forthe protein displayed on yeast cells or accordingto its inhibitory activity on Htt aggregation when


expressed as an intrabody inside living cells.15,17,49

Furthermore, using the SPOT technique, we wereable to narrow down the critical epitope to theminimal sequence EKLMKAFESLKSFQ. This stretchof residues is located just upstream of the Htt poly-Qregion that is critical for its oligomerization behavior.Nevertheless, the engineered VL domain effectivelyprevents Htt aggregation both in vitro and in vivo,especially during nucleation.10 This can be explainedby the crystal structure of the αHtt-VL/Htt5–18complex, which shows that the entire N-terminusof Htt is bound to the engineered Ig domain as alinear α-helix; hence, the αHtt-VL intrabody caninterfere with Htt aggregation via steric hindrance.Apart from that, αHtt-VL shields two of the three Lysresidues at the N-terminus of Htt, which impliesinterference with SUMOylation at these sites50 andpotentially indirectly affects Htt stability and/orlocalization.Unexpectedly, our structural analysis revealed

that the Htt peptide ligand is bound at the base ofthe CDRs (cf. Fig. 5) rather than among these loops,as it may be expected from camelid VHH domainsthat were crystallized in complex with antigens orlarge haptens.46,51 As a consequence of its α-helicalconformation, mainly side chains of the peptide arerecognized by αHtt-VL, which explains the highsequence specificity that became apparent from theepitope analysis. There was some indication duringthe epitope scanning experiments that Gln residuesat positions 19 and 20 might contribute to binding,although not part of the paratope in the crystalstructure, but this could also be due to neighboreffects (e.g., from the covalent attachment to thecellulose support).Notably, αHtt-VL appears to be thermodynami-

cally stable in the absence of a central disulfide bondas demonstrated by in vivo assays. However, afterreplacement of the Cys side chains, its affinity forHtt5–18 was decreased by 3 orders of magnitude invitro.17 Indeed, removal of the disulfide bond shouldbe expected to increase conformational flexibilitywithin αHtt-VL, thus entropically disfavoringHtt5–18binding. Subsequently, affinity maturation resultedin the identification of altogether four mutations(FL36I, YL50D, KL66R, and AL74T) that apparentlycompensate for the loss of the disulfide bridge.17These side-chain replacements can be grouped intofold-stabilizing and affinity-enhancing mutations.ArgL66 most likely improves the overall stability

of the αHtt-VL fold, as it forms hydrogen bonds withCDR-L1 and a salt bridge with AspL51 in CDR-L2,thus imposing conformational restraints on bothloops. Through its interaction with the CDR loops,this residue may also contribute to the stabilizationof the β-sandwich as a whole, by connecting β-sheets bed and gfcc′. In contrast, ThrL74 only seems toinfluence αHtt-VL stability in an indirect manner. Itslightly increases the overall surface hydrophilicity

of αHtt-VL but does not form relevant side-chaincontacts. On the other hand, both IleL36 and AspL50

form significant contacts with the bound Htt5–18peptide. AspL50 shows the largest conformationalchange upon epitope binding. It compensates thecharge of LysP6 and additionally contributes van derWaals contacts (cf. Table 2). However, AspL50 doesnot form hydrogen bonds to neighboring residues inαHtt-VL; consequently, its effect on fold stabilizationshould be marginal. IleL36 significantly contributesto the shape complementarity between αHtt-VL andHtt5–18. Its smaller size and more appropriate shapecompared with the original Phe side chain increasethe number of hydrophobic contacts.Usually, solitary human variable Ig domains are

prone to aggregation.45,52 However, in some cases,light chains or their variable domain fragments canform so-called Bence-Jones dimers,20 a class ofproteins found at elevated levels in the blood andurine of patients suffering from multiple myeloma.In this case, the hydrophobic interface, which isotherwise complemented by the heavy chain,37 isshielded by another light chain. Considering thehydrophobic surface properties of αHtt-VL (Fig. 6), itis obvious that although the number of nonpolarpatches at the interface is reduced, there are still twoprominent hydrophobic areas present around resi-dues TyrL49 and TrpL96. Notably, the Htt5–18 epitopepeptide is mainly bound via interactions involvingthese side chains. Thus, it seems that the highsolubility of αHtt-VL—compared with the presum-ably less soluble corresponding germline V1–11domain—arises by another mechanism: the engi-neered Ig domain assumes a pronounced overallnegative charge (Fig. 6), which correlates with itslow calculated pI around 4.6 (for residues 1–111).53

Apart from specialized animal Ig domains withantigen-binding activity, for example, from came-lids or sharks,45 human domain antibodies withdefined binding activities, so-called dAbs, have beenengineered.54 Because, in humans, VH or VLdomains are only found paired with their respectivecounterpart, the shielding of the hydrophobicinterface region is of utmost importance in theselection of soluble and stable dAbs from cloned V-gene libraries. Interestingly, some of these dAbs(which carry the conserved intradomain disulfidebond) still tend to form dimers of the Bence-Jonestype, at least in the crystallized state.55 While theαHtt-VL domain was also observed to form dimersin the crystal, these are clearly different from thiscanonical mode of V-domain pairing (Fig. 7): first,they involve the opposite β-sheet, that is, the leaf atthe back of the Ig domain, and second, theassociation is significantly different between theapo αHtt-VL and the αHtt-VL/Htt5–18 complex,showing that there is no conserved mode ofdimerization for this intrabody. Considering furtherthe biophysical observations from size-exclusion


chromatography and analytical ultracentrifugation,the engineered αHtt-VL domain shows a trulymonomeric state.Normally, intact antibodies, Fab fragments, scFv

fragments, and even single-domain antibodiesrecognize their epitopes exclusively via the CDRloops (Fig. 8). Generally, one should expect this wayof epitope recognition to be preserved duringantibody engineering. However, our structuralstudy demonstrates that binding modes differingfrom the classical scheme can be selected, leading tosimilar affinities. Comparison of theαHtt-VL/Htt5–18

(a)

(b)

αHtt-VL/Htt5-18

αHtt-VL/Htt5-18

αIGF-II antib

Fig. 8. Different ways of recognizing epitopes with α-helicaHtt5–18, the Fv fragment of an αIGF-II antibody (PDB entry 3Kdirected against human lysozyme (PDB entry 1OP9).57 VL andwhile CDRs are highlighted in light blue and light green. AntigeHtt5–18 and an FnIII-derivedMonobody in complexwith the hum2OCF), where an α-helix forms part of a more extended epitopethe central helix, we show αHtt-VL and the Monobody in the

structure with crystal structures of Nanobodies—and also of structurally related Monobodies/Adnectins58—suggests that the observed mecha-nism of epitope binding may not be entirely uniqueto αHtt-VL. Most strikingly, the structure of aMonobody based on the Ig-like fibronectin (Fn)type III domain and engineered to bind the humanestrogen receptor α ligand-binding domain59 in-teracts with an α-helix as central epitope element inan almost identical manner, involving the gfcc′ β-sheet (Fig. 8). Also, Monobodies directed against theAbelson Src homology 2 domain60 and the small

Monobody/hERα

VHH/lysozymeody

l structure by Ig domains. (a) From left to right: αHtt-VL/R3),56 and a camelid heavy chain antibody VHH domainVH domains are colored yellow and orange, respectively,ns are depicted in gray. (b) Comparison between αHtt-VL/an estrogen receptorα ligand-binding domain (PDB entry. To illustrate the almost identical mode of interaction withsame orientation [slightly tilted from (a)].


ubiquitin-related modifier61 exhibit energeticallyimportant contacts to the antigen via the β-sheet.Both in the Ig V-domain and in the FnIII fold, thelarge surface and the concave curvature of this β-sheet appear to be ideally suited to accommodate anα-helical peptide. In fact, targeted engineering of thisβ-sheet region may even appear as an alternativestrategy to generate antigen-specific Ig domainreagents.Taken together, the engineered αHtt-VL domain

reveals a previously unrecognized paratope forpeptide/antigen recognition arising from an inter-face area that—in conventional antibodies—isinvolved in the pairing with the VH domain. Thequestion arises whether this is a unique property ofthis particular Ig domain or it may represent a moregeneral mode of antigen recognition. In this regard,it has to be kept in mind that the original version ofαHtt-VL was discovered as an scFv fragment uponselection from a cloned human V-gene library builtfor yeast surface display.15 As no relevant addi-tional mutations emerged during the initial engi-neering of the isolated VL domain, it has to beassumed that the same mode of antigen binding didalready occur for the initially selected scFv. Thus,its VH and VL moieties must have dissociated inorder to allow binding of the peptide in thismanner, a conclusion that is also supported by thecomparable affinities of the single domain and thescFv.15 Given the generally weak associationbetween VL and VH domains of antibodies, evenwhen linked by a flexible peptide to form an scFvfragment, this hypothesis opens a new perspectiveon the results of many scFv selection experimentsreported so far. In fact, antigen recognition by justone of the variable domains alone—contrastingwith the intact combining site formed by the sixCDRs from both domains—might be a morefrequent phenomenon than anticipated up to now.

Materials and Methods

Vector construction and recombinant protein production

For bacterial production of αHtt-VL, two differentvectors were constructed, one encoding the engineeredVL domain with a C-terminal Strep-tag19 and one with aHis6-tag.

18 First, the coding region, starting with a Met-Gly-Ser leader sequence in front of the mature V-gene, wasamplified from pcDNA3.1Zeo(−)αHtt-VL12.3.217 usingphosphorothioate primers62 5′-CCC TCT CAT ATG GGTTCT CAG CCT GTG and 5′-GCT TAG GAC GGT GACCTT GGT and Turbo-Pfu DNA polymerase (Stratagene/Agilent, Santa Clara, CA). The PCR product was cut onone side with NdeI and cloned on pSA1-mDkk1, aderivative of the T7 promoter vector pRSET5a63 carryingalso the coding region for the Strep-tag, using NdeI andEco47III restriction sites. The resulting expression vector

was analyzed by restriction digest and DNA sequencingand dubbed pRSET-cVLstrep. From this plasmid, thecoding region for αHtt-VL was further subcloned onpASK75his, a derivative of the tetracycline promotervector pASK7564 carrying the coding region for the His6-tag, using XbaI and Eco47III restriction sites, resulting inthe expression vector pASK75-cVLhis.Recombinant protein production from pRSET-cVLstrep

was accomplished with the E. coli strain BL21(DE3)/pLysE.65 After growth in 2 L LB medium66 supplementedwith 100 mg/L ampicillin and 30 mg/L chloramphenicolin the shake flask at 22 °C for 8–25 h until OD550=0.4–0.9,gene expression was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside for 9–13 h or overnight. The cellswere harvested by centrifugation; resuspended in 20 mLof 100 mM Tris–HCl (pH 8.0), 150 mM NaCl, and 1 mMethylenediaminetetraacetic acid (EDTA); and lysed viaseveral passages through a French pressure cell(AMINCO, Rochester, NY). The cell extract was clearedby centrifugation (discarding part of αHtt-VL thatoccurred in aggregated form) and without dialysissubjected to affinity chromatography on Sepharose withimmobilized engineered streptavidin similarly as de-scribed before.19 The αHtt-VL-strep protein was firstdialyzed against gel-filtration buffer [10 mM Tris–HCl(pH 8.0); with or without 1 mM EDTA], then concentratedand finally purified by gel filtration on a Superdex 75column (Amersham Pharmacia, Uppsala, Sweden) in thepresence of the same buffer. The protein typically eluted intwo separate peaks appearing at variable volumes,together yielding up to 20 mg αHtt-VL-strep. Electrosprayionization mass spectrometry revealed a minor peak witha mass of 12,992 Da, which exactly corresponds to thecalculated mass for the full-length protein, and a majorpeak at 12,861 Da, indicating cleavage of the N-terminalMet residue.Recombinant protein production from pASK75-cVLhis

was achieved in the E. coli K-12 strain JM8367 in 2 L LBmedium supplemented with 100 mg/L ampicillin. In thiscase, gene expression was induced at OD550=0.5–0.6 for6–9 h by addition of 0.2 mg/L anhydrotetracycline.64 Afterharvest by centrifugation, the cells were resuspended in20 mL of 100 mM Tris–HCl (pH 8.0) and 1 mM EDTA andgently lysed by sonification, and then bacterial DNA wasdigested by addition of 2 mM MgCl2 and 30 U/mLBenzonase for 1–5 h at 4 °C. The cell extract was cleared bycentrifugation (again discarding some insoluble αHtt-VLprotein), and the supernatant was dialyzed against 50 mMHepes–NaOH (pH 7.5). After addition of solid glycinebetaine to a final concentration of 0.5 M, αHtt-VL-his waspurified by IMAC on Zn(II)-charged IDA-Sepharose(Amersham) in the presence of the same buffer.68 Elutionwas effected with an imidazole concentration gradientfrom 0 to 350 mM. After supplementation of theappropriate fraction with 10 mM EDTA and dialysisagainst gel-filtration buffer, the protein solution wasconcentrated with Amicon Ultracel 15 10,000-molecular-weight-cutoff ultracentrifugation units (Millipore, Billeri-ca, MA) and finally purified on a Superdex 75 gel-filtrationcolumn equilibrated with 10 mM Tris–HCl (pH 8.0)(sometimes containing 1 mM EDTA). αHtt-VL-his elutedin up to three peaks at variable elution volumes, yieldingup to 14 mg per 2 L of culture. The protein preparationswere finally dialyzed and concentrated using ultrafiltra-tion units as appropriate for the following experiments.


SPOT synthesis and binding assay

SPOT synthesis of arrays of variant epitope peptideswas performed on a Whatman 50 cellulose membrane(Whatman, Maidstone, UK) using an automated peptidesynthesizer (INTAVIS, Köln, Germany) according topublished procedures.22,69 Side-chain deprotection wasperformed according to the standard SPOT synthesisprotocol. Briefly, the peptides were synthesized on anamino-functionalized cellulose membrane as distinctspots. A β-alanine dipeptide spacer was inserted betweenthe membrane support and the C-terminus of eachpeptide. The peptide was extended in a stepwise mannerby using standard fluorenylmethoxycarbonyl solid-phasepeptide synthesis, followed by cleavage of the side-chainprotecting groups using trifluoroacetic acid. The designand the peptide spot arrangement for the substitutionaland length analyses of variant peptides derived from theMATLEKLMKAFESLKSFQQQ sequence were carried outusing an in-house software (LISA, version 1.71).After deprotection, the SPOT membrane was washed

once with ethanol and three times with Tris-buffered saline(TBS) buffer [137 mM NaCl, 2.7 mM KCl, and 50 mMTris–HCl (pH 8.0)] for 10 min each. Then the membranewas blocked and incubated for 3 h with 50 mL of asolution containing 5 mL blocking buffer (SIGMA-Aldrich,Steinheim, Germany), 2.5 g sucrose, and 5 mL 10×TBS.Subsequently, the membrane was incubated for 14 hat 4 °C with 10 μg/mL αHtt-VL-strep dissolved in thesame buffer. After washing three times with TBS, weincubated the membrane for 3 h at room temperaturewith StrepTactin-HRP (horseradish peroxidase) conjugate(IBA, Göttingen, Germany) diluted 1:5000 in the blockingsolution from above and washed three times with TBSagain. Signals were developed using a chemiluminescentsubstrate (UptiLightWBHRP; Uptima, Montlucon, France)and recorded on a Lumi-Imager (Roche Diagnostics,Penzberg, Germany).

SPR measurements

Real-time interaction analysis between αHtt-VL-strepand the synthetic epitope peptides was performed on aBIACORE-X instrument (BIACORE, Uppsala, Sweden) at20 °C in Hepes-buffered saline (HBS) [10 mM Hepes–HCl(pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.005% (v/v)surfactant P20]. Two sets of experiments were carried outin which either αHtt-VL-strep or the peptides werecovalently immobilized onto the sensor chip. Data wereanalyzed using the BIAevaluation 3.0 software from theinstrument manufacturer under the assumption of the 1:1Langmuir model and using both the simultaneous kineticsmodel and the steady-state equilibrium analysis.In the first instance, the VL domain was immobilized on

a CM5 sensor chip using the amine coupling procedureaccording to protocols by the instrument manufacturer.Glutathione S-transferase (SIGMA-Aldrich), whichproved not to bind to any of the peptides in the SPOTassay, was immobilized to the reference channel. Afterblocking of the chip surface, a dilution series (from 500 μMdown to 4 nM) of either one of the peptides EKLMKA-FESLKSFQ, EKLMKAFESLKSFQQ, or EKLMKA-FESLKSFQQQ in HBS was applied at a flow rate of15 μL/min. These peptides had been synthesized by solid-

phase synthesis on a TentaGel Sram resin (Rapp Polymere,Tübingen, Germany) and purified via HPLC on a C-18column, followed by mass spectrometric analysis.In the second setup, the peptide C-Ahx-EKLMKA-

FESLKSFQQQ (Ahx, aminohexanoic acid) was immobi-lized onto a CM5 sensor chip by the thiol couplingmethod. As a negative control, the peptide RRLIE-DAEYAARG-βA-C, which lacks the minimal recognitionmotif AxESLxxF identified in the SPOT substitutionalanalysis of MATLEKLMKAFESLKSFQQK, was similarlyimmobilized to the reference channel. A dilution series(from 250 nM down to 4 nM) of αHtt-VL-strep in HBS wasapplied at a flow rate of 2 μL/min to reach signalsaturation, followed by dissociation for 30 min.

Analytical ultracentrifugation

Sedimentation equilibrium experiments were per-formed in a Beckman Optima XL-I analytical ultracentri-fuge with Ti-60 rotor equipped with a UV/Vis and aninterference detection unit (Beckman, Fullerton, CA).Solutions of αHtt-VL-his at concentrations of 23 μM in0.01 M Tris–HCl (pH 8.0) were applied to six-sector cellswith a path length of 12 mm. The samples werecentrifuged at 20,000 rpm and 4 °C for 72 h untilequilibrium was reached, whereby the protein gradientwas measured by UV absorption at 280 nm. Data analysiswas carried out with Origin (Beckman) and KaleidaGraph(Synergy Software, Reading, PA) softwares as previouslydescribed70 using a measured density of 1.000 g/mL forthe solvent and an averaged standard value of 0.724 mL/gfor the partial specific volume of the protein.71

Crystallization, structure determination, and structureanalysis

αHtt-VL crystals were grown from a 9.4 mg/mLsolution of αHtt-VL-his [MGS-VL(1–111)-AH6] using thevapor diffusion technique. Hanging drops mixed from1.5 μL protein solution [in 10 mM Tris–HCl (pH 8.0)] and1 μl reservoir solution [1.2 M (NH4)2SO4, 0.2 M NaCl, and0.1 M Ches–NaOH (pH 9.8)] were equilibrated at 20 °Cagainst 0.5 mL reservoir solution on siliconized glasscoverslips, leading to crystals within 2 weeks.Soaking of the αHtt-VL crystals with the epitope peptide

did not result in proper complex formation. Therefore,αHtt-VL and the EKLMKAFESLKSFQ peptide (Htt5–18)were subjected to co-crystallization experiments. Htt5–18was dissolved in water and neutralized with 0.1 MNaOH,yielding a peptide concentration of 13.2 mM. A solution of11.3 mg/mL αHtt-VL-his in 10 mM Tris–HCl (pH 8.0) wasmixed with Htt5–18, leading to final concentrations of9.0 mg/mL αHtt-VL-his and 2.6 mM Htt5–18, correspond-ing to a 1:4 molar ratio. Crystals of the αHtt-VL/Htt5–18complex were grown at 20 °C in sitting drops using vapordiffusion by mixing 2 μL of the αHtt-VL-his/Htt5–18solution with 1 μL of the reservoir solution [10% (v/v)PEG 600 and 0.1 M K2HPO4/citrate (pH 4.1)] andequilibrating against 0.5 mL of the reservoir solution,resulting in crystals after 2 weeks.Crystals were harvested with nylon loops (Hampton

Research, Aliso Viejo, CA), cryo-protected, and flashcooled in a 100-K nitrogen stream (Oxford Cryosystems,


Oxford, UK). Cryo-protection of αHtt-VL and αHtt-VL/Htt5–18 crystals was achieved by supplementing thereservoir solution with 28.75% (v/v) glycerol and 17.5%(v/v) PEG 400, respectively. Diffraction data werecollected on a mar345 imaging plate detector (MarRe-search, Hamburg, Germany) using monochromatic Cu-Kα

radiation from an RU-300 rotating anode generator(Rigaku, Tokyo, Japan) equipped with Confocal Max-Flux Optics (Osmic, Troy, MI). Data reduction wasperformed with the XDS Package.72

αHtt-VL crystals diffracted to a resolution of 2.05 Å andbelonged to the space groupP41212with unit cell parametersof a=b=107.7 Å and c=50.6 Å (Table 1). The asymmetricunit contained two αHtt-VL molecules, corresponding to aMatthews coefficient of 2.85 Å3/Da. The αHtt-VL structurewas solved by molecular replacement with the programPHASER73 using the coordinates of a published VL domainas part of an scFv fragment (PDB code 1MFA).74 Modelbuilding was performed with Coot,75 followed byrestrained and TLS refinement using REFMAC5.5.76

Crystals of αHtt-VL/Htt5–18 diffracted to 2.60 Å resolu-tion and belonged to the space group P21 with unit cellparameters of a=53.1 Å, b=89.7 Å, c=95.0 Å, and β=99.5°(Table 1). The asymmetric unit contained eight αHtt-VL/Htt5–18 complexes, corresponding to a Matthews coeffi-cient of 1.92 Å3/Da. After solving the structure bymolecular replacement with PHASER using the refinedαHtt-VL structure from above, we built the Htt5–18 peptidemanually with Coot, followed by restrained and TLSrefinement with REFMAC5.5.Both structures were validated with Coot and

MolProbity.23 Secondary structure elementswere assignedwith DSSP.77 Molecular interfaces and contacts wereanalyzed with PISA33 and CONTACT.78 Structural super-positions were performed with SUPERPOSE,79 andmolecular graphics were prepared with PyMOL,80 whilesurface electrostatics were calculated with APBS.35

Deposited coordinates and recombinant DNA constructswere sequentially numbered from 1 to 111 correspondingto the mature αHtt-VL. However, to facilitate comparisonwith other published VL structures, we used Kabatnumbering to describe and discuss the crystal structuresthroughout the study.81 Kabat numbering of αHtt-VLresidues is indicated by an L preceding the residue number.Thus, residues L1–L9, L11–L27, L27A, L27B, L28–L95,L95A, L95B, L96–L106, L107A, and L108 correspond toresidues 1–9, 10–26, 27, 28, 29–96, 97, 98, 99–109, 110, and111 in the sequentially numbered coordinate files.

Accession numbers

Coordinates and structure factors for αHtt-VL and αHtt-VL/Htt5–18 have been deposited in the Research Colla-boratory for Structural Bioinformatics PDB with accessionnumbers 3LRG and 3LRH, respectively.

Acknowledgements

The authors wish to thank K. Richter and H. Stalz,both at Technische Universität München, for help in

the analytical ultracentrifugation and mass spec-trometry measurements, respectively. The Heredi-tary Disease Foundation is acknowledged forproviding graduate research support to D.W.C.

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