Proc. Nadl. Acad. Sci. USAVol. 88, pp. 4498-4502, May 1991Biochemistry

A systematic mutational analysis of hormone-binding determinantsin the human growth hormone receptor

(hormone-receptor interactions/protein-protein interactions/site-directed mutagenesis)

STEVEN H. BASS, MICHAEL G. MULKERRIN, AND JAMES A. WELLS*Department of Protein Engineering, Genentech, Inc., 460 Pt. San Bruno Boulevard, South San Francisco, CA 94080

Communicated by George R. Stark, January 31, 1991

ABSTRACT A mutational strategy is presented that al-lowed us to identify hormone-binding determinants in theextracellular portion of the human growth hormone receptor(hGHbp), a 238-residue protein with sequence homology to anumber of cytokine receptors. By systematically replacing sidechains with alanine we probed the importance of chargedresidues (49 total, typically located on the surface), aromaticresidues (9 total), and neighbors of these (26 total). The alaninesubstitutions that were most disruptive to hormone binding arelocated predominantly in four segments of a cysteine-richdomain in the hGHbp, and collectively they form a patch whenmapped upon a structural model proposed for cytokine recep-tors. Control experiments with monoclonal antibodies con-firmed that most of these alanine substitutions do not disruptthe overall antigenic structure of the hGHbp. This high-resolution functional analysis will complement structural stud-ies and provides a powerful basis for evaluating and engineer-ing the energetics of hormone-receptor interactions. More-over, the hormone-binding determinants identified here maybe similarly located in other, homologous, receptors.

Human growth hormone (hGH) is homologous to a largefamily of hormones that includes prolactins, placental lacto-gens, and proliferins (for review see ref. 1). Collectively,these hormones regulate a vast array of physiological effects,including growth, lactation, differentiation, and electrolytebalance (for reviews see refs. 2 and 3). These biologicaleffects begin with the binding of hormone to specific cellularreceptors. Systematic mutational studies have revealed func-tionally important residues in hGH for binding to the hGHreceptor (4-6). In contrast, virtually nothing is known of thehormone-binding determinants in the hGH receptor.The hGH receptor cloned from liver (7) consists of a single

polypeptide chain (620 residues total) containing an extra-cellular hormone-binding segment (246 residues), a singletransmembrane region (23 residues), and a cytoplasmic seg-ment (351 residues). The extracellular portion of the hGHreceptor is found naturally in the blood stream (8) as anhGH-binding protein (hGHbp). Recent comparative se-quence analyses suggest that the hGHbp is structurallyrelated to a large family of cytokine receptors (9-11).The hGHbp (containing residues 1-238) has been ex-

pressed in Escherichia coli in large quantities (12), and itretains the same high affinity for hGH as its natural glyco-sylated counterpart. Here, using a combination of mutationaland biophysical analysis, we have identified important hor-mone-binding determinants in the hGHbp. These determi-nants are chemically complementary to those previouslyidentified in hGH, and they map predominantly to a cysteine-rich region of the receptor and to loop regions on one side ofa structural model proposed for cytokine receptors (10). This

mutational strategy should be applicable to probing otherhormone-receptor and protein-protein interactions forwhich little or no structural information is available.

MATERIALS AND METHODSRestriction enzymes, DNA polymerase I large fragment, T7DNA polymerase, T4 DNA polymerase, and polynucleotidekinase were from New England Biolabs, Bethesda ResearchLaboratories, or United States Biochemical. Genentech pro-vided hGH, synthetic oligonucleotides, and monoclonal an-tibodies (mAbs), except mAbs 5 and 265, which were pur-chased from Agen Biomedical (Parsippany, NJ).The plasmid phGHbp(1-238) (ref. 12), coding for residues

1-238 of the wild-type hGHbp sequence (7), contains the florigin to allow preparation of single-stranded DNA for mu-tagenesis (13) and sequencing (14). Mutants of the hGHbpwere expressed in E. coli and purified as described for thewild-type hGHbp (12). Briefly, cultures of E. coli KS330 (15)carrying plasmid phGHbp(1-238) secreted properly foldedand processed hGHbp into the periplasm. Periplasmic pro-teins were released from the cells by a freeze-thaw andextraction into hypotonic buffer (10 mM Tris HCI, pH 7.5)containing 2 mM EDTA and 1 mM phenylmethylsulfonylfluoride to inhibit metallo- and serine proteases, respectively.The hGHbp was precipitated with ammonium sulfate (45%saturation), resuspended in the same buffer, and clarified bycentrifugation. At this point the affinity of each hGHbpmutant for hGH was determined. Controls show that con-taminating proteins from E. coli do not interfere with the hGHbinding assay (5). Mutant binding proteins with decreasedbinding affinity to hGH were purified to homogeneity aspreviously described (12) except for W104A, which waspurified by Q-Sepharose chromatography (Pharmacia) fol-lowed by a mono-Q column (Pharmacia).

Purified binding proteins were dialyzed against 10 mMTris HCI, pH 7.5/100 mM NaCI for circular dichroic spectra.Protein concentrations were determined from the absorbancespectrum using A`1t° = 2.35 for hGHbp (12) and AO-'% = 2.11for W104A and W104F. Spectra were obtained on an AvivAssociates (Lakewood, NJ) Cary 60 spectropolarimeter inthe near-UV (320-250 nm) at a 0.5-nm interval and in thefar-UV (250-190 nm) at a 0.2-nm interval. All spectra wereaveraged over 5 scans with a 2-sec averaging time for eachdatum.

Binding constants were determined from competitive dis-placement assays using 125I-labeled hGH as a tracer (16). Ananti-receptor mAb (mnAb 263; ref. 17) and 15% (vol/vol)

Abbreviations: hGH, human growth hormone; hGHbp, humangrowth hormone-binding protein; mAb, monoclonal antibody. Singlemutants are designated by the wild-type residue (single-letter aminoacid code) followed by its sequence position and the mutant residue.For example, F96S indicates a mutation in which Phe-% is convertedto Ser.*To whom reprint requests should be addressed.

4498

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

7, 2

020

Proc. Natl. Acad. Sci. USA 88 (1991) 4499

polyethylene glycol were used to precipitate the bindingprotein hormone complex after equilibration overnight at250C (12). Under these conditions hGH and the hGHbp forma one-to-one complex.

RESULTSBinding of Natural Variants of the hGHbp to hGH. North-

ern analysis and cDNA cloning from human hepatocytesproducing the hGH receptor revealed a messenger RNA thatis lacking the exon 3 sequence encoding residues 7-28 (ref.18; W. Wood, personal communication). We constructed abinding protein missing residues 7-28. This protein has thesame affinity for 125I-labeled hGH as does full-length hGHbp(Table 1), suggesting these amino-terminal residues are notessential for hGH binding.Goossens and coworkers (19) sequenced the coding region

of the hGH receptor from one family with Laron-type dwarf-ism and found a single phenylalanine-to-serine substitution atposition 96 (F96S) in the extracellular domain of the hGHreceptor. They postulated this mutation resulted in produc-tion of a receptor defective for hGH binding. We producedthe F96S mutant hGHbp in E. coli and found it to have a Kdfor hGH that was identical to that of the wild-type hGHbp(ref. 20; Table 1), suggesting that this defect does not affecthGH binding directly.

Scanning-Mutational Analysis of Charge Clusters. To de-termine if charged residues of the hGHbp are involved inhGH binding, site-directed mutagenesis was used to replacesystematically all arginine, lysine, aspartic, and glutamicresidues with alanine. We substituted charged side chainswith alanine because in the absence of other structuralinformation we anticipated charged residues to be exposed tosolvent and the alanine substitutions to cause minimal per-turbation in the folding of the protein. Alanine, the mostcommon amino acid (21), is small and is found in both buriedand exposed positions in proteins (for reviews see refs. 22 and23).The charged residues were mutated in clusters of 2-5

residues to maximize the efficiency of our analysis. Fourteenclustered charge-to-alanine mutants were prepared that col-lectively mutated all of the charged residues (except Lys-110)from position 31 to 217 in the hGHbp (Table 1). Eight ofthesemultiple mutants expressed sufficient quantities in E. coli tobe analyzed, and two of them (RRE 70-75 and KDKEE203-209) showed reductions in binding affinity to hGH ofabout 6-fold.We next dissected the six nonexpressing and two disrup-

tive binding variants by producing single-alanine mutants (30total) in these segments (Table 1). Ofthese mutants, only one(D85A) could not be expressed in quantities that allowedanalysis. Nine of the single-alanine mutants, E42A, E44A,R70A, R71A, E82A, D126A, E127A, D132A, and K215A,caused reductions in binding affinity to hGH of 2- to 8-fold.The disruptive effects in the RRE 70-75 mutant can beaccounted for by R70A and R71A. In contrast, dissection ofthe pentamutant KDKEE 203-209 did not single out a singleresidue prominent in disrupting binding, although slight ef-fects were observed for K203A and K206A. We interpretthese data to mean that side chains in this charged segmentare not substantially involved in binding to hGH.

Alanine-Scanning of Tryptophans and Tyrosines in the Cys-teine-Rich Domain. Fluorescence experiments show thatbinding of hGH to the hGHbp causes changes in the tryp-tophan fluorescence spectra of the hGHbp (M.G.M., unpub-lished results). The charged-to-alanine-scan data indicatethat the strongest binding interactions with hGH are locatedin the cysteine-rich region of the hGHbp (Fig. 1). Of the fourtryptophan residues in this region (Trp-50, Trp-76, Trp-80,and Trp-104), only the WSOA mutant could not be expressed

Table 1. Dissociation constants for binding of mutants of thehGHbp to hGH

hGHbpmutant

Natural variantsWild typeA7-28*F96S

Clustered charge-to-Ala scantKEKK 31-37RERE 39-44DEK 52-59RRE 70-75EKE 79-82DE 85-91DEK 119-121DED 126-132DRE 152-158RDK 161-167EEKE 173-180EKKD 183-190KDKEE 203-209RRKR 211-217

Charge-to-Ala scanR39AE42AR43AE44AR70AR71AE75AE79AK81AE82AD85AE91AK11OAD126AE127AD132AE173AE175AK179AE180AK203AD205AK206AE207AE209AR211AR213AK215AK217AE224A

Kd, nM

0.40 ± 0.050.42 ± 0.110.32 ± 0.05

0.60 ± 0.04NE

0.56 ± 0.132.03 ± 0.10

NENE

0.57 ± 0.25NE

0.68 ± 0.130.36 ± 0.06

NE0.49 ± 0.152.65 ± 1.07

NE

0.63 ± 0.033.20 ± 0.010.64 ± 0.041.20 ± 0.130.81 ± 0.101.10 ± 0.040.35 ± 0.040.35 ± 0.040.51 ± 0.051.50 ± 0.09

NE0.54 ± 0.060.43 ± 0.012.10 ± 0.370.80 ± 0.152.50 ± 0.080.46 ± 0.140.35 ± 0.140.40 ± 0.020.56 ± 0.050.60 ± 0.200.37 ± 0.120.56 ± 0.020.35 ± 0.040.36 ± 0.040.44 ± 0.090.29 ± 0.060.93 ± 0.140.70 ± 0.030.48 ± 0.01

hGHbpmutant Kd, nM

Scan ofTrp andTyrWSOA NEW76A 1.03 ± 0.18W80A 0.53 ± 0.08W104A >1000W104F 44 ± 0.4Y68A NEY86A NEY9SA 0.56 ± 0.06Y107A NE

Scan nearchargedand TrpdeterminantsS40A NEP41A 0.62 ± 0.12T45A NEF46A NES47A 0.39 ± 0.07

T5lAt 0.45 ± 0.06

N72AT73AQ74AT77AQ78A

F96AN97AS98AS99AF100ATlOlAS102A1103AI1OSAP106A

S124AV125A1128AV129AQ130A

0.19 ± 0.060.19 ± 0.040.15 ± 0.030.26 ± 0.030.20 ± 0.03

NE0.26 ± 0.040.23 ± 0.040.17 ± 0.020.39 ± 0.022.5 ± 0.17

0.88 ± 0.020.83 ± 0.070.50 ± 0.1433.7 ± 16

NE2.75 ± 0.041.08 ± 0.130.37 ± 0.010.38 ± 0.05

Mutants were prepared by site-directed mutagenesis (13), ex-pressed in E. coli, and assayed as described in Materials andMethods. Results are presented as mean ± SEM. NE, not expressedin levels suitable for analysis.*A7-28 indicates a deletion mutant of exon 3 in which residues fromThr-7 to Asn-28 were deleted.tThe KEKK 31-37 mutant, for example, is a tetraalanine mutant inwhich Lys-31, Glu-32, Lys-34, and Lys-37 have all been mutated toAla. This nomenclature applies to all of the clustered charge-to-alanine variants.tTaken from ref. 12.

in amounts sufficient for binding analysis (Table 1). TheW80A mutant caused no significant reduction in bindingaffinity, whereas W76A caused a 3-fold reduction in affinity.

Biochemistry: Bass et al.

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

7, 2

020

Proc. Natl. Acad. Sci. USA 88 (1991)

8-6

-D6-E (D 4 -oI

c 2-

10 20 30 40 e 5 50 60 70

8-

-D( -E- 4 -

sCc-02-

E0Ic:E I0 -§

WKECPDYVSAGENSCYFNSSFTSIWIPYCIKLTSNGGT'VDEKC FSVDEIVOPDPPIALNWTLLNVSLTGIHADIOVRWE80 Fe 100 110 S- 130 140 150

84-6

2-APRNADIOKGWMVLEYELOYKEVNETKWKMMDPILTTSVPVYSLKVDKEYEVRVRSKORNSGNYGEFSEVLYVTLPOMSO160 170 180 190 200 210 220 230

Residue

In contrast, we could not detect any binding to the W104Amutant (reduced more than 2500-fold) and a more conserva-tive variant (W104F) was reduced in binding by 110-fold.These reductions in binding affinity for mutating Trp-104 areby far the largest in the entire set.Such a large change in binding could be a result of

structural change in hGHbp. To assess the structural effectsof these mutants we determined the circular dichroic (CD)spectra for the mutants W104A and W104F and wild-typehGHbp (Fig. 2). Both the near- and the far-UV CD spectra aresimilar for each of these proteins. A comparison of thenear-UV CD spectra of the wild type with the spectra of bothmutants indicates that the substitution of the tryptophanresults in virtually no change except for an expected decreasein the intensity of the tryptophan spectrum. The far-UV CDshows some alterations in intensity but the overall shapeappears to be the same for each of the proteins, indicatingthey are not grossly misfolded.

lodination of the hGHbp disrupts binding to hGH (J.A.W.,unpublished results). We therefore mutated the four ty-rosines to alanine in the cysteine-rich domain includingTyr-68, Tyr-86, Tyr-95, and Tyr-107. However, only Y95Acould be expressed in amounts sufficient for analysis, and ityielded a dissociation constant essentially the same as that ofthe wild-type hGHbp (Table 1). Although Tyr-95 does notappear to be involved in binding to hGH, we do not knowabout the importance of the others.

Alanine-Scanning of Residues Flanking Disruptive Chargedand Tryptophan Residues. Protein-protein interfaces oftencontain several binding segments each contributing morethan one side-chain determinant of hydrophobic or hydro-philic character (for reviews see refs. 24 and 25). Thus, wereplaced with alanine the neutral and hydrophobic residuesthat closely flank those charged and tryptophan residuesfound to be most disruptive to binding (Table 1). Within thefirst disulfide loop no additional residues were identifiedbeyond Glu-42 and Glu-44 that disrupted binding; however,we were unable to express S40A, T45A, and F46A in quan-tities suitable for analysis. In the region around Arg-70 andTrp-76, no other disruptive alanine mutants were identified.However, we identified four mutants, N72A, T73A, Q74A,

FIG. 1. Histogram showing the effectupon binding to hGH for alanine substi-tutions scanned over the hGHbp (resi-dues 31-224). Values of Kd (mutant)/Kd(wild-type hGHbp) were calculated fromdata in Table 1. Sites of alanine substi-tutions causing effects ranging from a2-fold to >2500-fold decrease in affinitycluster in four segments in the cysteine-rich domain. The disulfide pairings(-S-S-) are taken from Fuh et al. (12).Single alanine replacements are indicatedby single bars, and clustered alanine var-iants and the A7-28 mutant are indicatedby linked bars.

and Q78A, that slightly increased affinity for hGH. Theregion flanking Trp-104 contained four other residues, Thr-101, Ser-102, Ile-103, and Pro-106, where alanine substitu-tions caused a loss in binding affinity of 2- to 85-fold, and one(S99A) that slightly increased affinity, about 2-fold. Finally,the region from Asp-126 to Asp-132 contained two additionaldisruptive alanine mutants, V125A and I128A.

Binding of mAbs to Mutants of hGHbp. To evaluate thestructural integrity of hGHbp mutants that were reduced inaffinity for hGH, we analyzed their binding properties with apanel of six different anti-hGHbp mAbs, whose affinitiesranged from about 0.5 to 6 nM (Table 2). These mAbs wereprepared by immunizing with the natural rabbit or rat, growthhormone receptors (mAb 5, ref. 26; and mAb 263, ref. 17), or

Cf)O

x

0

-2

-4

-6

-8

50

0

3: -50

2 -100

-150

-200

260 270 280 290 300 310

Wavelength (nm)

FIG. 2. CD spectra of wild-type hGHbp ( ) and the mutantsW104A (-.-----) and W104F ( ). CD spectra were obtained on

solutions of -1 mg/ml in 10 mM Tris-HCI, pH 7.5/100 mM NaCl at20°C. (A) Far-UV CD spectra of proteins in a 0.01-cm cell. (B)Near-UV CD spectra in a 1.0-cm cell. The units of mean residueellipticity, [O]MRw, are degrees cm2-decimole-1.

- -I I

>2,500-*jj -84

Al-L

hA

2200 210 220 230 240

1 -----------A-N w --------0-1 ---- m -----I---------

I - ----- 11 ----------------a -----OMM-1 ---------- A ------II0v

4500 Biochemistry: Bass et al.

DI

Iac

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

7, 2

020

Proc. Natl. Acad. Sci. USA 88 (1991) 4501

Table 2. EC50 for binding of six anti-hGHbp mAbs to the wild-type and variants of the hGHbp that disrupt bindingof hGH

mAb EC50, nM

hGHbp mutant 5 263 3B7 3D9 12B8 13E1Wild type 0.7 ± 0.2 0.6 ± 0.1 0.5 ± 0.4 2.2 ± 1.0 6.1 ± 1.8 3.2 ± 1.7E42A 0.5 ± 0.1 0.5 ± 0.3 0.2 ± 0 1.7 ± 0.1 2.5 ± 1.8 0.8 ± 0.3E44A 1.7 ± 1.0 0.7 ± 0.3 0.8 ± 0.3 3.3 ± 2.2 3.3 ± 0.9 2.7 ± 0.5E82A 1.0 ± 0.6 5.3 ± 1.9 0.7 ± 0.4 2.9 ± 2.1 1.5 ± 0.4 0.9 ± 0.2T1OlA 1.7 ± 0.4 0.7 ± 0.06 >100 >100 14.0 ± 2.1 8.2 ± 2.0W104A 1.3 ± 0.3 0.6 ± 0.07 0.7 ± 0.07 2.2 ± 0.07 5.4 ± 1.6 3.1 ± 0.6P106A 2.8 ± 0.8 0.8 ± 0.4 1.0 ± 0.2 8.5 ± 0.9 33.4 ± 21 7.8 ± 0.3V125A 0.8 ± 0.2 0.4 ± 0.06 0.5 ± 0.1 3.2 ± 1.2 2.4 ± 0.3 0.9 ± 0.3D126A 1.0 ± 0.1 0.6 ± 0.1 0.7 ± 0.1 3.9 ± 1.6 3.9 ± 0.4 1.7 ± 0.91128A 1.4 ± 0.1 0.4 ± 0.1 0.7 ± 0.1 6.4 ± 0.8 5.7 ± 0.7 0.9 ± 0.1D132A 1.5 ± 0.5 0.7 ± 0.06 0.6 ± 0.06 4.2 ± 1.8 15.5 ± 7.4 5.1 ± 1.9BlockshGH binding No No No No Yes Yes

An ELISA format was used in which microtiter plates were coated with mAb, then incubated with 3-fold serial dilutionsofthe purified hGHbp mutant. The extent ofbinding was quantified by incubating with purified rabbit polyclonal anti-hGHbpantibody, then a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Results are mean ± SEM.

with the native hGHbp expressed and purified from E. coli(mAbs 3B7, 3D9, 12B8, and 13E1).Most of the hGHbp mutants reacted with all of the mAbs

as well as the wild-type hGHbp (Table 2). However, theTlOlA variant was reduced in binding by >200- and 45-foldto mAbs 3B7 and 3D9, respectively, and by about 2-fold withthree out of the remaining four mAbs. Similarly, the bindingaffinity ofthe P106A mutant to the same five mAbs is loweredby 2- to 5-fold. It is unlikely that all of these effects aremediated by direct effects that the TiOlA and P106A muta-tions have on the antibody binding epitopes. Typically, directeffects show up as a highly selective disruption pattern (4, 5).For example, the E82A variant is reduced in binding affinityto mAb 263 by 9-fold, yet it is virtually unchanged in bindingto the other five mAbs. Of the mAbs tested 13E1 and 12B8completely block binding ofhGH to the hGHbp. The fact thatmost of the alanine mutants that disrupt receptor binding donot disrupt binding of the mAbs that block hGH binding isconsistent with these being overlapping but nonidentical, aswas typically seen for hGH (4, 5).

DISCUSSIONWe have presented a scheme to systematically identifyhormone-binding determinants where little structural infor-mation was available. This analysis has identified four seg-ments located in the cysteine-rich domain of the hGHbp thatare important to binding of hGH (Fig. 1). In order of decreas-ing importance to binding these segments include residuesThr-101 to Pro-106 >> Val-125 to Asp-132 > Arg-70 toGlu-82 Glu-42 to Glu-44. The segments Thr-101 to Pro-106and Val-125 to Asp-132 should be in close proximity becausethey are bridged by the Cys-108 Cys-122 disulfide.Recent sequence analyses suggest that the extracellular

binding domains of the growth hormone and prolactin recep-tors are structurally homologous to a large number of cytok-ine and other receptors including the interleukin (IL)-2, IL-3,IL-4, IL-6, IL-7, erythropoietin, and granulocyte-macroph-age colony-stimulating factor (GM-CSF) receptors (refs.9-11 and references therein). Bazan (10) predicted that theextracellular portions of these receptors contain two immu-noglobin-like domains, each consisting of seven P-strandsand connecting loops. When the alanine mutants that causea 2-fold or greater effect upon binding to hGH are placed uponthe topographic map predicted for the cysteine-rich domainof the hGHbp (Fig. 3) they form a patch in the four loops atone end of the model. By comparison, residues altered away

from these putative loop regions have virtually no effect uponbinding.Our data do not support the hormone-binding site that was

predicted (10) to span the G-,8 strand of the N-terminaldomain (residues 119-125) and the D-,3 strand in the C-ter-minal domain (residues 184-195). Moreover, the CD spectra(Fig. 2) do not substantiate the extensive (3-sheet structurepredicted for the hGHbp. The far-UV CD spectrum (Fig. 2A)is similar to the spectrum of the kringle of tissue plasminogenactivator (27), which is composed mainly of turns or loopsthat are held together by disulfide crosslinks. The CD spec-trum of the hGHbp is different from that of the T-cellreceptor, CD4 (28), which has a (-sandwich type fold exhib-iting -50%,p-sheet structure overall (29, 30). However, wenote that p-sheet content is more difficult to quantify accu-rately than a-helix by CD measurements and that the aro-

P - Q~P7

.P T N y

s 100; F .

40 .S F. ,S ,Q F Y. F

R S .S ,E L C-S-S-C

C -S-S- C N W80 Q K

K H K K 110 ,E 120

T W 50oY P L .

F T C -S-S- C G T V

K D S P L S T

P E N :D~.,N 60 N> G- - _. . G

3' K 4.EE.,H Y- T

go Go ..

A S

A B E D C F G

FIG. 3. Location of residues in the hGHbp causing a 2-fold orgreater effect upon binding to hGH mapped upon an immunoglob-ulin-like folding diagram predicted for the cysteine-rich domain (10).Seven antiparallel 13-strands (A-G) compose a ,3-sandwich by foldingthe G strand around the back until it hydrogen bonds to the A strand.Residues causing less than a 2-fold reduction (0), a 2- to 4-foldreduction (0), a 4- to 10-fold reduction (0), a 10- to 100-foldreduction (0), and >100-fold reduction (*) in binding are indicated.Those residues that gave low expression yields are indicated by C).Disulfides are shown by-S-S- connecting cysteine residues, andresidues are numbered by tens.

Biochemistry: Bass et al.

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

7, 2

020

Proc. Natl. Acad. Sci. USA 88 (1991)

matic residues and disulfides can contribute substantially tothe far-UV CD spectrum. Thus, our data are consistent withthe positioning of the loops in the structure predicted forcytokine receptors (10) but cannot confirm the degree of,a-sheet structure predicted.

Several lines of evidence suggest that the changes inbinding affinity for the hGHbp mutants are not a result of theproteins being grossly misfolded. First, these proteins can beexpressed at levels comparable to wild-type hGHbp. Poorexpression can be correlated with an unstable or misfoldedprotein (31). Second, mAb binding to most of the mutantproteins parallels the binding found for wild-type hGHbp. Wedo not know that the mAbs raised to native hGHbp react onlywith the native hGHbp, because denatured or reducedhGHbp aggregates in solution (data not shown). However,structural analyses of antibody-antigen complexes (for re-view see ref. 32) and epitope mapping of hGH (4) and CD4(29, 30) show that virtually all of these epitopes are highlydiscontinuous. Third, the CD spectra in both the near- andfar-UV of the W104A and the W104F mutant are similar to thespectrum of wild-type hGH (Fig. 2). The fact that the W104Fmutation is reduced in affinity by 100-fold, whereas theW104A mutant is reduced >2500-fold, demonstrates the keenrequirement for a large hydrophobic side chain at position 104for tight binding to hGH. Although we do not believe themutations cause enormous changes in the folding of themutant proteins, it is possible that for some of these mutants(perhaps T1OlA or P106A) the reduction in binding affinityresults from indirect structural effects as opposed to makingdirect side-chain contacts with hGH. It is notable that neitherof these later mutants cause substantial changes in the CDspectra (data not shown), suggesting the CD in this case isless sensitive than the mAb binding analysis.The combined mutational analyses of hGH (5) and the

hGHbp show there is a great deal of functional complemen-tarity at the interface. Of the 10 charged-to-alanine substitu-tions in the hGHbp causing a 2-fold or greater reduction inhormone binding affinity, 7 are acidic residues; any one ofthetop 5 of these (E42A, E44A, E82A, D126A, and D132A) ismore disruptive than any of the three disruptive basic resi-dues (R70A, R71A, and K215A). By comparison, alanine-scanning mutagenesis of hGH has revealed that 3 of the top5 disruptive binding mutants are basic residues (R64A,K172A, R178A) and none are acidic residues. Thus, there iselectrostatic complementarity between an electropositivebinding epitope on hGH and an electronegative epitope onthe hGHbp. Moreover, both epitopes contain importanthydrophobic determinants as well.The distribution of strong and weak binding determinants

is not identical between the epitope on hGH as compared tothe hGHbp. For example, of the 17 disruptive alanine mu-tants in the hGHbp, 10 cause 2- to 4-fold reductions inbinding, 5 cause 4- to 10-fold reductions, 1 residue causes a10- to 100-fold disruption, and 1 causes greater than 1000-foldreduction in affinity for hGH. By comparison, of the 20disruptive alanine mutants in hGH, 8 cause 2- to 4-foldreductions, 7 cause 4- to 10-fold reductions, 5 cause 10- to100-fold reductions, and none cause over 100-fold reductionin binding to the hGHbp (5, 6). These data suggest that thereis not a simple one-to-one side-chain to side-chain interactionbetween hGH and the hGHbp.

In conclusion, the mutational analyses provide a functionalmap of side chains in the hGHbp important for binding ofhGH. We cannot be sure that additional side-chain bindingdeterminants are present because not all residues weretested. A high-resolution structure of the hormone-receptorcomplex will be necessary to determine if these residues

modulate binding by direct or indirect effects and to definemain-chain interactions. The functional data provided here,when combined with high-resolution structural information,should provide a solid basis from which one may begin torationally design small-molecule mimics of hGH.

We are grateful to Dr. Brian Fendley and Marcie Winget forproviding mAbs; to Peter Ng, Parkash Jhurani, and Mark Vasser forsynthesis of oligonucleotides; to Dr. Brad Snedecor, Mike Brochier,and Michael Covarrubias for fermentations; to Dr. William Wood forhelpful suggestions; and to Wayne Anstine for graphics.

1. Nicoll, C. S., Mayer, G. L. & Russell, S. M. (1986) Endocrine Rev.7, 169-203.

2. Chawla, R. K., Parks, J. S. & Rudman, D. (1983) Annu. Rev. Med.34, 519-547.

3. Isaksson, O., Eclen, S. & Jansson, J. 0. (1985) Annu. Rev. Physiol.47, 483-499.

4. Cunningham, B. C., Jhurani, P., Ng, P. & Wells, J. A. (1989)Science 243, 1330-1335.

5. Cunningham, B. C. & Wells, J. A. (1989) Science 244, 1081-1085.6. Cunningham, B. C., Henner, D. J. & Wells, J. A. (1990) Science

247, 1461-1465.7. Leung, D. W., Spencer, S. A., Cachianes, G., Hammonds, R. G.,

Collins, C., Henzel, W. J., Barnard, R., Waters, M. J. & Wood,W. I. (1987) Nature (London) 330, 537-543.

8. Baumann, G., Stolar, M. W., Ambara, K., Barsano, C. P. &DeVries, B. C. (1986) J. Clin. Endocrinol. Metab. 62, 134-141.

9. Cosman, D., Lyman, S. D., Idzerda, R. L., Beckmann, M. P.,Park, L. S., Goodwin, R. G. & March, C. J. (1990) Trends Bio-chem. Sci. 15, 265-270.

10. Bazan, J. F. (1990) Proc. Natl. Acad. Sci. USA 87, 6934-6938.11. Patthy, L. (1990) Cell 61, 13-19.12. Fuh, G., Mulkerrin, M. G., Bass, S., McFarland, N., Brochier, M.,

Bourell, J. H., Light, D. R. & Wells, J. A. (1990) J. Biol. Chem.265, 3111-3115.

13. Kunkel, T. A., Roberts, J. D. & Zakour, R. A. (1987) MethodsEnzymol. 154, 367-382.

14. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad.Sci. USA 74, 5463-5467.

15. Strauch, K. L. & Beckwith, J. (1988) Proc. Nat!. Acad. Sci. USA85, 1576-1580.

16. Spencer, S. A., Hammonds, R. G., Henzel, W. J., Rodriguez, H.,Waters, M. J. & Wood, W. I. (1988) J. Biol. Chem. 263,7862-7867.

17. Barnard, R., Bundesen, P. G., Rylatt, D. B. & Waters, M. J. (1985)Biochem. J. 231, 459-468.

18. Godowski, P. J., Leung, D. W., Meacham, L. R., Galgani, J. P.,Hellmiss, R., Keret, R., Rotwein, P. S., Parks, J. S., Laron, Z. &Wood, W. I. (1989) Proc. Natl. Acad. Sci. USA 86, 8083-8087.

19. Amselem, S., Duquesnoy, P., Attrec, O., Novelli, G., Bousnina, S.,Postel-Vinay, M.-C. & Goossens, M. (1989) N. Engl. J. Med. 321,989-995.

20. Bass, S. & Wells, J. A. (1990) N. Engl. J. Med. 322, 834.21. Klapper, M. H. (1977) Biochem. Biophys. Res. Commun. 78, 1018-

1024.22. Chothia, C. (1976) J. Mol. Biol. 105, 1-14.23. Rose, G. D., Geselowitz, A. R., Lesser, G. L., Lee, R. H. &

Zehfus, M. H. (1985) Science 229, 834-838.24. Argos, P. (1988) Protein Eng. 2, 101-113.25. Janin, J., Miller, S. & Chothia, C. (1988) J. Mol. Biol. 204,155-164.26. Barnard, R., Bundeson, P. G., Rylatt, D. B. & Waters, M. J. (1984)

Endocrinology 115, 1805-1813.27. Cleary, S., Mulkerrin, M. G. & Kelley, R. F. (1989) Biochemistry

28, 1884-1891.28. Chamow, S. M., Peers, D. H., Bryon, R. A., Mulkerrin, M. G.,

Harris, R. J., Wang, W.-C., Bjorkman, P. J., Capon, D. J. &Ashkenazi, A. (1990) Biochemistry 29, 9885-9891.

29. Wang, J., Yan, Y., Garrett, T. P. J., Liu, J., Rodgers, D. W.,Garlick, R. L., Tarr, G. E., Husain, Y., Reinherz, E. L. & Harri-son, S. C. (1990) Nature (London) 348, 411-418.

30. Ryu, S.-E., Kwong, P. D., Truneh, A., Porter, T. G., Arthos, J.,Rosenberg, M., Dai, X., Xuong, N., Axel, R., Sweet, R. &Hendrickson, W. A. (1990) Nature (London) 348, 419-426.

31. Pakula, A. A., Young, V. B. & Sauer, R. T. (1986) Proc. Natl.Acad. Sci. USA 83, 8829-8833.

32. Davies, D. A., Padlan, E. A. & Sheriff, S. (1990) Annu. Rev.Biochem. 59, 439-473.

4502 Biochemistry: Bass et al.

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

7, 2

020