J. Mol. Biol. (1990) 215, 175-182
`
`Framework Residue 71 is a Major Determinant of the
`Position and Conformation of the Second
`Region in the Va Domains
`Hypervariable
`of Immunoglobulins
`
`Anna Tramontano1, Cyrus Chothia2·3 and Arthur M. Lesk1·2
`
`1 European Molecular Biology Laboratory
`M eyerhof strasse I
`Postfach 1022.09
`6900 Heidelberg, F.R.G.
`2 M RC Laboratory of Molecular Biology
`Cambridge, CB2 2QH, U.K.
`3Christopher Ingold Laboratory
`University College London
`20 Gordon Street
`London WCIH OAJ, U.K.
`
`(Received 2 January 1990; accepted 18 May 1990)
`
`Analysis of the immunoglobulins of known structure reveals systematic differences in the
`
`
`
`position and main-chain conformation of the second hypervariable region of the VH domain
`
`of the position of H2 is the size of the residue at
`(H2). We show that the major determinant
`
`site 7 I, a site that is in the conserved framework of the V H domain. It is likely that for a.bout
`two thirds of the known V H sequences the size of the residue at this site is also a major
`of H2. This effect
`
`determinant of the conformation
`
`can override the predisposition of the
`sequence, as in the case of the H2 loop of J539, which is a.n exception to the rules relating
`
`
`sequence and conformation of short hairpin loops. Understanding the relationship between
`71 and the position and conformation of H2 has applications
`the residue at position
`to the
`
`
`
`prediction and engineering of antigen-binding sites of immunoglobulins.
`
`high range of specificity achieved by antibodies (Wu
`
`
`
`& Kabat, 1970; Ka.bat et al., 1987).
`1. Introduction
`
`Immunoglobulins a.re multi-domain proteins
`
`The atomic structures of several immunoglobulin
`
`consisting of two chains, a. light cha.in with one
`fragments have been determined by X-ray crystal­
`variable ( VLt) and one constant domain, and a
`lography (Davies & Metzger, 1983; Alza.ri et al.,
`
`heavy chain containing one variable domain (VH)
`1988). They show that all the domains have a very
`and three constant domains. The antigen-binding
`similar folding pattern: two P-sheets pa.eked face to
`site is formed by six loops, three from the VL and
`
`face. A core of the double P-sheet structure, ca.lied
`three from the VH domains. Figure 1 shows a simpli­
`
`the framework, has a very similar conformation in
`
`fied view of the antigen-binding site, indicating the
`
`different variable domains because of the conserva­
`
`
`
`relative positions of the loops. The variability of the
`
`tion of internal residues and the requirements of
`
`residues in the antigen-binding site gives rise to the
`
`internal pa.eking. The residues that form the inter­
`face between the VL and VH domains a.re also
`strongly conserved.
`These results have led to the view that the frame­
`
`work structure plays an essentially passive role in
`
`the structural variation that occurs in the a.ntigen­
`binding site.
`175
`
`t Abbreviations used: VH, variable dome.in of
`immunoglobulin heavy chains; VL, variable dome.in of
`immunoglobulin light chains; H2, second hyperve.rie.ble
`loop of heavy cha.in; r.m.s., root-mean-square; cxa, right­
`handed ex-helical conformation; cxt_, left-handed a-helical
`conformation.
`
`0022-2836/90/170175--08 $03.00/0
`
`<g 1990 Academic Press Limited
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`176
`
`A. Tramontano et al.
`
`Figure 2 shows the genera.I structural context of
`H2 within the VH domain.
`
`2. Co-ordinates and Calculations
`Protein structures used in this work are listed in
`Table I. The atomic co-ordinates of these structures
`are distributed by
`the Protein Data Bank
`(Bernstein et al., 1977), except for the refined co­
`ordinates of J539 which are a private communica­
`tion from Drs E. A. Padlan and D. R. Davies. The
`structures were displayed using Insight (Dayringer
`et al., 1986) on an Evans & Sutherland PS390.
`Programs written by A.M.L. (Lesk, 1986) were used
`for analysis of
`the structures and database
`searching.
`Throughout the paper residue numbers refer to
`the heavy-chain numbering scheme of Kabat et al.
`(198'7). In VH domains, the conserved P-sheet frame­
`work consists of residues 3 to 12, 17 to 25, 33 to 52,
`56 to 60, 68 to 82, 88 to 95 and 102 to 112 (Chothia
`& Lesk, 1987). These residues were used in the
`superpositions of VH domains.
`
`3. The Conformations of H2 Loops
`In VH sequences the second hypervariable region
`consists of a P-hairpin, comprising residues 50
`through 65 (Wu & Kabat, 1970; Kabat et al., 1987).
`In the known VH structures the main-chain confor­
`mations of residues 50 to 52 and 56 to 65 are the
`same: for high-resolution well-refined structures the
`backbone atoms of residues 50 to 52 and 56 to 64 fit
`with a root-mean-square (r.m.s.) deviation between
`0·4 and 0·7 A. This region is illustrated in Figure 3:
`
`Figure 1. Outline structure of the antigen-binding site.
`The site is formed by 6 loops of polypeptide (.l'N) linked
`to strands in P-sheets (0).
`
`In the immunoglobulins of known structure the
`conformations of the second hypervariable region in
`VH (H2) differ. The position of the H2 with respect
`to the conserved framework is also variable. For
`example, in the VH domains of immunoglobulins
`J539 and HyHEL-5, the H2 regions have the same
`number of residues. If the framework structures are
`superimposed, the CP atoms in residue 53, at the tip
`of H2, are found to differ in position by 6-3 A
`(1 A=O·l nm). Here, we show that the variations in
`two structural features of H2, its position and its
`conformation, are coupled, and that they depend in
`large part on the nature of the amino acid residue
`that occupies position 71
`in the heavy-chain
`framework.
`
`Hl
`
`H2
`
`Hl
`
`H2
`
`Figure 2. The structure.I context of H2 within the VH dome.in of Fa.b J539. H2 is shaded relatively darkly, Hl is
`shaded relatively lightly. The thick broken circle indica.tes the g1uw1idinium group of Arg371.
`
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`Position a:n.d Conformation of the H2 Loop
`
`177
`
`I mmunoglobulin heavy chain variable domains of known atomic structure
`
`Table 1
`
`Molecule
`
`NEWM
`HyHEL-10
`
`HyHEL-5
`KOL
`J539
`
`H2 sequence
`
`Residue 71
`
`Reference
`
`y H G
`y s G
`
`p G s G
`D D G
`s
`p D s G
`
`v
`R
`
`A
`R
`R
`
`Saul et al. ( 1978)
`Padlan et al. (1989)
`
`Sheriff et al. (1987)
`Marquart et al. ( 1980}
`Suh et al. (1986)
`
`N K G N K y
`McPC603
`R
`Satow et al. (1986)
`4-4-20
`N K p y N y
`R
`Herron et al. ( 1989)
`The H2 residues are those between positions 52 and 56 (see text).
`
`the main-chain atoms of residues 56 to 60 form
`hydrogen bonds to those of residues 48 to 52 to form
`a P-hairpin. Sequence variations in these residues
`have little or no effect on the main-chain conforma­
`tion, because the side-chains are on the surface. The
`turn that
`links these two strands, comprising
`residues 52a to 55 or 53 to 55, we refer to as the H2
`region. In the known structures it differs in length
`and conformation.
`Hairpin structures have been classified according
`to their length and conformation (Venkatachalam,
`1968; Efimov, 1986; Sibanda & Thornton, 1985;
`Sibanda et al., 1989). Particular conformations are
`usually associated with characteristic sequence
`patterns. The positions of Gly, Asn, Asp and Pro
`residues are important because these residues allow
`main-chain conformations that in other residues
`cause strain.
`
`(a) Three-residue H2 regions
`In NEWM and HyHEL-10, the H2 loop is a
`three-residue hairpin, residues 53 to 55_ The NEWM
`H2 loop is shown as conformation I in Figure 3. The
`usual sequence requirement for this conformation is
`a Gly (or Asn or Asp) at the third position (residue
`55), which can take up a++ conformation (that is,
`
`</>>0, l/1>0) (Sibanda et al., 1989). Both NEWM and
`HyHEL-10 have a glycine at this position:
`
`NEWM
`HyHEL-10
`
`53
`
`Tyr
`Tyr
`
`His
`Ser
`
`55
`
`Gly
`Gly
`
`71
`
`Val
`Arg
`
`and in both cases the Gly is in a + + conformation.
`
`(b) Four-residue H2 regions
`loop of HyHEL-5
`is a four-residue
`The H2
`hairpin, residues 52a to 55. This is shown as. confor­
`mation 2 in Figure 3. The conformation is close to
`the one most commonly observed in four-residue
`turns, in which the first three residues are in an cxR
`conformation and the fourth in an cxL conformation.
`These turns normally require Gly in the fourth
`position (Efimov, 1986; Sibanda & Thornton, 1985;
`Sibanda et al., 1989), as observed in HyHEL-5.
`The H2 regions in KOL and J539 form four­
`residue turns with a conformation different from
`HyHEL-5. They both have the third residue (54) in
`the cxL conformation and the first, second and fourth
`in the cxR conformation. This is shown as conforma­
`tion 3 in Figure 3.
`
`�
`��-··
`L .....
`50 <\._ .. ... .. .....
`
`.
`
`I
`3
`KOL
`01·3
`NEWM
`J539
`HyHEL-10
`NOIO
`Figure 3. The main-chain conformations of the 2nd hypervariable region in V H domains in the immunoglobuline of
`known structure. The conformations are numbered l to 4. The immunoglobulins in which these conformations a.re found
`are listed under each number.
`
`4
`McPC603
`4-4-20
`
`2
`HyHEL-5
`NC41
`
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`178
`
`A. Tramontano et al.
`
`Table 2
`Results of a database search for main-chain conformations the same as that of the H2
`loop of KOL
`
`!J.
`(Al
`
`0-18
`0-19
`0-22
`0-22
`0-22
`0·23
`0·24
`0·28
`0-29
`0-29
`
`Molecule (Protein Data. llank code)
`
`Rhizopuspepsin (3APR)
`Subtilisin Carlsberg (2SEC)
`Ribonuclease A (7RSA)
`Pepsinogen (lPSG)
`434 repressor protein ( 1 R69)
`Calmodulin (3CL,.�)
`Calmodulin (3Ck�)
`Adenylate kinase (3ADK)
`Fab J539
`Cyt-0chrome c551 (451C)
`
`Starting
`residue
`
`145
`10
`32
`142
`35
`57
`21
`166
`353
`9
`
`Sequence
`
`s Q G
`L
`I K A
`L
`s R N
`L
`D Q G
`L
`E N G K
`A D G N
`K D G D
`I
`K R G
`p D s G
`N K G c
`
`!J., root-mean-square deviation ofN, <::', C and 0 atoms of residues 53 to 56 of the l'H domain of KOL
`and well-fitting nlglons from other known structures.
`
`This type of turn has not been described
`previously, but we find that it occurs fairly often in
`proteins. We searched the database of solved struc­
`tures for regions similar in main-chain conformation
`to the H2 loop of KOL. Table 2 lists the closest
`matches: ten loops, including J539 H2, for which
`the r.m.s. difference in position of main-chain atoms
`is less than 0·3 A. There are 61 such loops with
`r.m.s. deviation less than 0·5 A. For KOL and J539
`H2 and the nine best-fitting non-homologous loops,
`the average values of the conformational angles and
`their standard deviations are:
`
`(see Fig. 5(b)). The r.m.s. deviation of all N, C", C
`and 0 atoms is 0·96 A. The McPC603 H2 loop is
`shown as conformation 4 in Figure 3. The sequences
`in these regions a.re:
`
`52&
`
`52b
`
`52c
`
`53 54
`
`55
`
`71
`
`McPC603
`4-4-20
`
`Asn Lys Gly Asn Lys Tyr Arg
`Asn Lys Pro Tyr Asn Tyr Arg
`
`In both structures residue 54 is in the aL confor­
`mation. In the other VH sequences with six-residue
`
`Angle
`Mean (deg.)
`Stand.ard deviation (deg.)
`
`4'2
`1/11
`4'1
`-61 -35 -95
`12
`12
`8
`
`1/12
`77
`14
`
`efi3
`65
`11
`
`"''
`q,,
`1/13
`22 -78 -18
`12
`13
`11
`
`Of the nine loops in Table 2, excluding J539, seven
`have a Gly in the third position, like KOL, one has
`Asn and one has Lys. Of all the loops with r.m.s.
`deviation less than 0·5 A, none is like J539 in having
`Gly at only the fourth position.
`These results show that H2 in J539 is an excep­
`tion to the rules relating sequence and structure in
`short hairpins. Both HyHEL-5 and J539 have Gly
`in the fourth position of the loop:
`
`KOL
`J539
`HyHElr5
`
`52&
`
`Asp
`Pro
`Pro
`
`53
`
`Asp
`Asp
`Gly
`
`54
`
`Gly
`Ser
`Ser
`
`55
`
`Ser
`Gly
`Gly
`
`The position of Gly in J539 should imply a confor­
`mation of H2 similar to that of HyHEL-5. Instead
`the conformation observed in J539 is the same as in
`KOL (see Fig. 4(b) and Fig. 5(a)). The r.m.s. devia­
`tion in the position of the H2 main-chain a.toms in
`J539 and HyHEL-5 is I·9 A; for J539 and KOL it is
`0-3 A. The residues of H2 in J539 make no non­
`bonded contacts to residues other than those in Hl
`and Arg7l and Asn73 (see Fig. 2).
`
`(c) Six-residue H2 regions
`In McPC603 and 4-4-20, the H2 loops are six­
`residue hairpins. Their conformations a.re similar
`
`H2 loops, the residues found at this position are
`Gly, Asn or Asp (Ka.bat et al., 1987). It is interesting
`to note in this context that the Lys at position 54 in
`McPC603 is the result of a somatic mutation from a
`germ-line gene that contains a. Gly.
`
`The Interactions of H2 with the Framework
`Examination of the interactions of the H2 loops
`with the rest of the V H domain shows that the
`determinants of the conformations of four-residue
`H2 foops are not entirely within the sequence of the
`loop itself, but involve the packing of the loop
`against the rest of the structure.
`In Figures 4 and 5 we show, for pairs of anti­
`bodies of known structure, the relative positions of
`the HI and H2 loops and the contacts made by
`certain side-chains. The relative positions of these
`loops in these Figures are those induced by the
`superposition of the framework structures. The
`Figures show that the HI loops occupy rather
`similar positions with respect to the framework in
`alJ the known structures. But the positions of the
`H2 loops a.re in some cases very different. These
`differences are related to the size of the residue at
`position 71.
`KOL and J539 have four-residue H2 loops in very
`similar positions and conformations (Fig. 4(b)). The
`residue at position 71 is Arg in both structures. The
`
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`

`Position a:n.d Conformation of the H2 Loop
`
`179
`
`H2
`
`~ ·
`··i
`·· .. )
`'\) Phe 29/lle 29
`
`Arg 71/ Val 71
`
`(a)
`
`( b)
`Figure 4. The relative positions of the H l and H2 hypervariable regions and of framework residue 71, in different pairs
`of immunoglobulins. The Hl and H2 regions are represented by their c• atoms. The positions shown here are those found
`after the superposition of the V H framework residues (see text). (a) NEWM (continuous lines) and HyHEL-10 (broken
`lines). (b) KOL (continuous lines) and J539 (broken lines).
`
`side-chains of these a.rgmme residues are buried.
`They form hydrogen bonds to main-chain atoms of
`residues in the HI and H2 loops and pack against
`the Phe at position 29.
`The superposition of J539 and HyHEL-5 shown
`in Figure 5(a.) illustrates the case of two immuno­
`globulin structures with H2 loops of the same length
`but different conformation and position. In J539, in
`which residue 71 is an Arg, residue Pro52a. in the H2
`loop is on the surface. In HyHEL-5, in which
`residue 7l is an Ala, Pro52a is buried, filling the
`cavity that would be created by the absence of a
`long side-chain at position 71. The manner in which
`these H2 loops pa.ck against the rest of the VH
`domain explains why the H2 region of J539 does not
`have the conformation that we would expect from
`Gly at position 56. If it did have the expected
`conformation, like that in HyHEL-5, the Pro52a.
`side-chain would occupy the same space as the aide­
`chain of Arg71 (Fig. 5(&)). The set of torsion angles
`
`that move the side-chain of Pro52a !I-Way from
`Arg71 require an H2 conformation different from
`that in HyHEL-5.
`In both McPC603 and 4-4-20, H2 is a six-residue
`turn, and residue 71 is an Arg. In McPC603 Arg7 l
`has its side-chain buried, and is hydrogen bonded to
`the main-chain of HI and H2, as in KOL and J539
`(Fig. 5(b)). The Tyr at the sixth position (55) packs
`against Arg71.
`
`5. The Role of Residue 71
`These observations can be summarized as follows.
`(I) Position 71 contains a small or medium-sized
`residue. For three and four-residue H2 loops the
`residue at position 53/52a packs against residues at
`positions 71 and 29. This brings the HI and H2
`loops close together and puts four-residue H2 loops
`in conformation 2 (Fig. 3).
`(2) Residue 71 is &n arginine. The side-chain of
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`180
`
`A. Tramontano et al.
`
`Pro 520
`
`H2
`
`............
`
`HI
`
`'
`
`A'"
`
`Alo 71
`
`(a)
`
`(b)
`Figure S. The relative positions of the Hl and H2 hypervariable regions and of framework residue 71, in pairs of
`known immunoglobulin structures. The Hl and H2 regions a.re represented by their C" a.toms. The positions shown here
`are those found after the superp osition of the VH framework residues (see text). (a) HyHEL-5 (continuous Jines) and
`J539 (broken lines); (b) McPC603 (continuous lines) and 4-4-20 (br<>ken lines).
`
`the argmme is buried between HI and H2, and
`forms hydrogen bonds with the main-chain in both
`loops. The H2 loop is displaced from HI with
`residue 52a. on the surface. Four-residue H2 loops
`have conformation 3 (Fig. 3).
`In Fab NC41 residue 71 is a Leu, intermediate in
`size. In
`the structure of
`the Fab NC41-
`(Colman et al., 1987;
`neuraminidase complex
`Chothia et al., 1989), H2 has the HyHEL-5 confor­
`mation. Residue 52a in NC41 is a Thr, smaller than
`the Pro at the corresponding position in HyHEL-5;
`as a result the shift in H2 produced by the Leu is
`reduced.
`For six-residue H2 loops we have information for
`McPC603 and 4-4-20, in which residue 71 is Arg. All
`known VH sequences that contain six-residue H2
`loops have Arg a.t position 71 (see below).
`
`6. Applications to Structure Prediction. The H2
`Regions in ImmunoglobuJins of
`Unknown Structure
`
`To see if the results reported here are useful for
`predicting the structures of antigen-binding sites of
`immunoglobulins we must find out whether the
`determinants of the known conformations are
`commonly present in sequences of VH domains of
`unknown structure. Kabat e.t al. (1987) have
`collected the known immunoglobulin sequences. We
`found in this collection 302 V H sequences for which
`all, or almost all, residues in the region 50 to 75 are
`known. Of these sequences, 54 are from humans and
`248 are from mice.
`There are 47 sequences with three-residue H2
`regions. All these have Gly or Asp at position 55.
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`Position and Conformation of the H2 Loop
`
`181
`
`This implies that they have conformations similar
`to that of H2 in NEWM and HyHEL-10: an impli­
`cation supported by the prediction of the conforma­
`tion of the H2 region in Dl.3 (Chothia et al., 1986).
`At position 71, Arg or Lys occurs in 43 sequences
`and Val or Leu in four.
`There are 194 sequences with four-residue H2
`regions. Of these, 35 sequences have Arg or Lys at
`position 7I and Gly, Asn or Asp at position 54. For
`these we have the clear expectation that the R2
`regions have conformation 3 of Figure 3 and a
`position close to that found in KOL and J539.
`Another 99 sequences have Pro at position 52a; Gly,
`or in a few cases Asn or Asp, at position 55; and Val,
`Leu or Ala. at 71. Again we have the clear expec­
`tation these domains have H2 loops in conformation
`2 of Figure 3 and in a position
`like that of
`HyHEL-5.
`Most of the 41 other four-residue H2 regions do
`not have a Gly, Asn or Asp at either position 54 or
`55. The expectation that these have conformations
`like that in KOL/J539 or HyHEL-5, depending on
`the residue at position 71, is more tentative. The
`structure of Fab NQIO has recently been deter­
`mined (Spinelli et al., unpublished results). In NQlO,
`the sequence of H2 is S-G-S-S, with Arg in position
`71 (Berek et al., 1985). The occurrence of Gly at the
`second position of a four-residue hairpin is very
`unusual; it does not occur in any of the loops
`surveyed by Chothia. & Lesk (1987) and Sibanda et
`al. (1989). (KOL has GJy at the third position of the
`loop; and HyHEL-5 has Gly at the fourth position
`of the loop). The conformation and position ofH2 in
`NQIO are the same as in KOL: The -r.m.s. deviation
`of all N, C", C and 0 atoms of H2 is 0·39 A; the
`r.m.s. deviation of all N, C", C and 0 atoms of HI
`and H2 together is 0·43 A (Chothia et al., I989). This
`then confirms the importance of the residue at
`position 71 in determining the conformation of the
`loop in these cases.
`There are 61 sequences with six-residue H2
`regions. All have Tyr at position 55; Arg at 71 and
`all but two have Gly, Asn or Asp at 54. The conser­
`vation at these sites suggests these H2 regions have
`conformations close to that in McPC603 and 4-4-20.
`
`7. Applications to Antibody Engineering
`The ability to transplant hypervariable regions of
`non-human origin to human frameworks
`is of
`medical importance (Reichmann et al., I 988). For
`the binding site of the synthetic product to be the
`same as that in the original antibody, the frame­
`works should have the same residues at those sites
`important for the positions and conformations of
`the hypervariable regions. However, binding sites
`do have a. limited intrinsic flexibility. The main­
`cha.in portions of close-packed segments of proteins
`can move relative to each other by 1 to 2 A, with
`little expenditure of energy (Chothia. et al., 1983),
`and the a.pices of loops may well be able to move by
`larger amounts . Thus the effect on antigen binding
`of changing the conformation, or the position and
`
`orientation, of a hypervaria.ble region will depend
`upon whether the region is involved in binding and,
`if it is, on how much energy is required for the
`structural readjustments necessary to form the
`correct interactions. The most serious effects will
`occur when the framework contains a large residue,
`rather than a small one, as compression energies are
`large.
`Jones et al. (1986) transplanted the antigen­
`binding loops from the heavy chain of a mouse
`antibody on to the framework of a human one. They
`observed that the synthetic product, when bound to
`the original mouse light chain, had the same affinity
`for the hapten as the original mouse antibody.
`Inspection of the two sequences used by Jones et al.
`(1986) shows that both the mouse and human anti­
`bodies have Va.I at position 71, and therefore we
`should expect the four-residue H2 loop from the
`mouse antibody to retain its conformation and posi­
`tion on transfer to the human framework.
`Verhoeyen et al. (1988) transferred the hyper­
`varia.ble regions of the heavy chain of the mouse
`anti-Iysozyme antibody Dl.3 to the framework of
`the human antibody NEWM. An affinity for lyso­
`zyme was retained, although reduced approxi­
`mately tenfold. Both DI . 3 and NEWM contain a
`Gly at position 55 of the heavy chain; at position 71
`DI .3 contains Lys and NEWM contains Val. This
`would suggest that in the synthetic antibody H2
`has the correct conformation but is displaced from
`the position in DI .3. In the DI .3-lysozyme complex,
`the contacts ma.de by H2 (residues 53 to 55) to the
`antigen involve residues Gly53 and Asp54 (Amit et
`al., 1986). We cannot determine to what extent the
`slight loss of affinity by the synthetic antibody is
`associated with
`the molecular
`readjustments
`requiired to retain these contacts.
`Reichmann et al. (I988) reshaped an antibody by
`transplanting all six hypervariable regions from a
`rat antibody on to a human framework for both VL
`and VH domains. In this case H2 had six residues, as
`does McPC603. The parent rat antibody has Arg at
`position 71, but the human framework has Val.
`There is no known VH sequence with the combina­
`tion a. six-residue H2 and Val at position 71 (see
`above). The synthetic antibody has an affinity close
`to that of the rat original. Whether this is because
`the cavity created by the smaller residue does not
`significantly affect the conformation of the six­
`residue H2, or because this H2 makes only a
`marginal contribution to affinity, is unclear.
`
`8. Conclusion
`Previously we reported that framework residues
`are an important determinant of the conformation
`of first hyperva.ria.ble region of V L (Lesk & Chothia,
`I982; Chothia & Lesk, 1987). In that case the nature
`of the framework residues is related directly to the
`class of the light cha.in: " or A.. The analysis
`presented here demonstrates that a. framework
`residue plays a major role in determining position
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`182
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`A. Tramontano et al.
`
`and conformation of a hypervariable region within
`one class of domains, the VH.
`We have also found a clear exception to the rules
`that relate the sequences and conformations of
`hairpin loops. The H2 region in J539 adopts a
`conformation stabilized by tertiary
`interactions
`that override the predisposition of its sequence
`pattern. A prediction of the conformation of this
`loop, based only on the local sequence, would be
`incorrect.
`The results reported here will help in under­
`standing the molecular mechanisms involved in the
`generation of antibody diversity, extend the rules
`governing sequence-structure correlations in short
`hairpins, and, together with our previous analysis of
`the other hypervariable regions (Chothia & Lesk,
`1987), improve the accuracy of predicted immuno­
`globulin structures.
`
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`Edited by A. Fersht
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