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`Journal of Molecular Biology
`
`Editor-in-Chief
`
`P. Wright
`Department of Molecular Biology, Research Institute of Scripps Clinic
`10666 N. Torrey Pines Road, La Jolla, CA 92037, USA.
`
`Assistant Editor
`J. Karn
`MRC Laboratory of Molecular Biology
`Hills Road, Cambridge CB? QQH, U.K.
`
`Founding Editor
`Sir John Kendrew
`
`Consulting Editor
`Sydney Brenner
`
`Editors
`
`P. Chambon, Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Institut de Chimie Biologique.
`Faculté de Médecine, 11 Rue Humann, 67085 Strasbourg Cedex, France.
`A. R. Fersht, University Chemical Laboratory, Cambridge University, Lensfield Road, Cambridge CB2 lEW, U.K.
`M. Goltesman, Institute of Cancer Research, College of Physicians & Surgeons of Columbia University,
`701 W. 168th Street, New York, NY 10032, USA.
`'
`P. van Hippel, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403—1229, USA.
`It. Huber, Max-Planck-Institut fiir Biochemie, 8033 Martinsried bei Miinchen, Germany.
`A. Klug, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 ZQH. U,K_
`
`Associate Editors
`
`’
`
`C. R. Cantor, Human Genome Center, Donner Laboratory, Lawrence Berkeley Laboratory, University of California
`Berkeley, CA 94720, USA.
`'
`N.-H. Ohua, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA,
`I". E. Cohen, Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco,
`CA 94143—0446, USA.
`I). J. Deltosier, Rosenstiel Basic Medical Sciences Research Center. Brandeis University, Waltham, MA 02254. USA.
`W. A. Hendrickson, Department of Biochemistry 8L Molecular Biophysics, College of Physicians & Surgeons of
`Columbia University, 630 West 168th Street, New York, NY 10032, USA.
`1.13. Holland,
`Institute de Genetique et Microbiologie, Batiment 409, Université de Paris X1. 91405 ()rsay Cedex 05,
`France.
`‘
`B. Honig, Department of Biochemistry & Molecular Biophysics, College of Physicians & Surgeons of Columbia
`University, 630 West 168th Street, New York, NY 10032, USA.
`V. Luzzati, Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, 91 Cif—slu‘.Yvettex France.
`J. L. Mandel, Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Institut de Chimie Biologique,
`Faculté de Médecine, 11 Rue Humann, 67085 Strasbourg Cedex, France.
`B. Matthews, Institute of Molecular Biology, University of Oregon, Eugene, 0R197403—1229, USA.
`J. H , Miller, Department of Microbiology, University of California, 405 Hilgard Avenue, Los Angeles, CA 90024. USA.
`M. F. Moody, School of Pharmacy, University of London, 29/39 Brunswick Square, London WCIN lAX, UK.
`T. Richmond, Institut fiir Molekularbiologie und Biophysik, Eidgenlissische Technische Hochschule. Honggerberg,
`CH 8093 Zurich, Switzerland.
`R, Schleif, Biology Department, Johns Hopkins University. Charles & 34th Streets. Baltimore, MD 21218, USA.
`N. L. Sternberg, Central Research & Development Department, E.
`I. du Pont Nenlours & Company, \Vilmington,
`DE 19898, USA.
`K. R. Yamamoto, Department of Biochemistry and Biophysics, School of Medicine, University of California.
`San Francisco, CA 94143—0448, USA.
`M. Yanagida, Department of Biophysics, Faculty of Science, Kyoto University, Sakyo-Ku, Kyoto 606, Japan.
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`
`

`

`
`
`176 A. Tramontano et a1.
`
`
`
`‘ Figure-1. ()utline structure of the antigen-binding site.
`'l he site is formed by 6 loops of polypeptide (IW) linked
`to strands in [i-sheets ([1).
`E
`
`In the immunoglobulins of known structure the
`conformations of the second hypervariable region in
`VH (H2) difi'er. The position of the H2 with respect
`to the conserved framework is also variable. For
`example,
`in the VH domains of immunoglobulins
`.1539 and HyHEL-S, the H2 regions have the same
`nu m her of residues. If the framework structures are
`superimposed, the C” atoms in residue 53, at the tip
`of H2, are found to difTer
`in position by 63111
`(1 r :0] nm). Here. we show that the variations in
`two'strmmural 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.
`
`Figure 2 shows the general structural context of
`H2 within the VH domain.
`
`2. Co-ordinates and Calculations
`
`Protein structures used in this work are listed in
`Table 1. The atomic co—ordinates of these structures
`
`by the Protein Data Bank
`are distributed
`(Bernstein et al., 1977), except for the refined co—
`ordinates 0f 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 P8390.
`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.
`(1987). In VH domains, the conserved fi-sheet frame-
`work consists of residues 3 to l2, 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 [i-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 07 A. This region is illustrated in Figure 3:
`
`
`
`Figure 2. The structural context of H2 within the VH domain of Fab [1539. H2 is shaded relatively darkly, H1 is
`shaded relatively lightly. The thick broken circle indicates the guanidinium group of Arg37l.
`
`

`

`
`
`Position and Conformation of the H2 Loop 177
`
`Iin'rn’a'noglobul in heavy chain variable domains of known atomic structure
`
`
`Molecule
`H2 sequence
`Residue 71
`Reference
`
`
`Table 1
`
`NEWM
`HyHEL-IO
`
`HyH Eli-5
`KOL
`.1539
`
`Y
`Y
`
`1’
`1)
`P
`
`H
`S
`
`1
`l)
`1)
`
`G
`(.1
`
`S
`G
`S
`
`l
`S
`l
`
`V
`It
`
`A
`R
`R
`
`Saul er a]. (1978)
`Padlan of.
`(1.1. (1989)
`
`Sheriff cl ((1. (1987)
`Marquart e! a]. (1980)
`Suh et at. (1986)
`
`Satow et (21. (1986)
`R
`V
`K
`N
`G
`K
`N
`MOPU603
`
`
`
`
`
`
`
`
`N K P Y N Y R44-20 Herron et al. (1989)
`
`The H2 residues are those between positions 52 and 56 (see text).
`
`gb>0, tfl>0) (Sibanda et ai., 1989). Both NEWM and
`HyHEL—IO have a glycine at this position:
`
`
`
`
`
` 53 54 55 7|
`
`
`
`the main—chain atoms of residues 56 to 60 form
`
`hydrogen bonds to those of residues 48 to 52 to form
`a B-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 at, 1989). Particular conformations are
`usually associated with characteristic sequence
`patterns. The positions of (11y, Asn, Asp and Pro
`residues are important because these residues allow
`main-chain conformations that
`in other residues
`cause strain.
`
`(a) ’l’lm‘ee—residae [1.9 regions
`
`the H2 loop is a
`In NEWM. and HyHEL-IO,
`three-residue hairpin, residues 53 to 55. The NEWM
`H2 loop is shown as conformation 1 in Figure 3. The
`usual sequence requirement for this coi‘iformation is
`a Gly (01' Asn or Asp) at the third position (residue
`55), which can take up a + + conformation (that is,
`
`Val
`(11y
`His
`’l‘yr
`NEWM
`
`l-lyHEL- 1 0 Arg Tyr Scr (11y
`
`
`
`
`and in both cases the Gly is in a + + conformation.
`
`(b) Ii‘ou‘r—residue [[2 regions
`
`loop of HyH Eli-5 is a four—residue
`The 1—12
`hairpin, residues 528. 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 ocR
`conformation and the fourth in an (XL conformation.
`These turns normally require (11y in the fourth
`position (Efimov, 1986; Sibanda & Thornton, 1985;
`Sibanda et al., 1989), as observed in HyHEL—5.
`The H2 regions in KO L and .1539 form foqu
`residue turns with a conformation different from
`
`HyHEL-5. They both have the third residue (54) in
`the (XL conformation and the first, second and fourth
`in the (IR conformation. This is shown as conforma—
`tion 3 in Figure 3.
`
`
`
`D|-3
`NEWM
`HyHEL—IO
`
`HyHEL-5
`NC4|
`
`KOL
`J539
`NQIO
`
`McPC 603
`4 — 4 _ 20
`
`Figure 3. The main-chain conformations of the 2nd hy}.)ervariable region in VH domains in the immunoglobulins of
`known structure. The conformations are numbered 1 to 4. The nnimnioglolnlhns in which these conformations are found
`are listed under each number.
`
`

`

`
`
`178 A. Tramontano et a1.
`
`Table 2
`
`Results of a database search for main-chain conformatzons the same as that of the H2
`
`loop of K0L
`
`A
`Starting
`
`(A)
`Molecule (Protein Data Bank code)
`residue
`Sequence
`
`L
`Jr
`Q
`S
`145
`Rliizopuspepsin (ISAPR)
`0-18
`A
`K
`1
`L
`10
`Subtilisin Carlsberg (2SEC)
`0'19
`L
`N
`R
`S
`32
`Ribonuclease A (7RSA)
`0-22
`L
`n‘.
`Q
`D
`H2
`Pepsinogen (IPSG)
`022
`K
`G
`N
`E
`35
`434 repressor protein (ll-€69)
`0'22
`N
`G
`D
`A
`57
`()almodulin (3(JLN)
`023
`D
`}
`D
`K
`21
`(Talmodulin (3(3LN)
`024
`I
`G
`R
`K
`166
`Adenylate kinase (3A1’)K)
`0'28
`G
`S
`D
`P
`353
`Fab J 539
`029
`
`
`
`
`
`
`Cytochrome 6551 (4510) 9 N K G029 C
`
`A, root-mean-square deviation of N, C“, (I and 0 atoms of residues 53 to 56 of the VH domain of KOL
`and well-fitting regions from other known structures
`
`turn has not been described
`type of
`This
`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. Table2 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,
`In both structures residue 54 is in the (XL conform
`the average values of the conformational angles and
`mation. In the other VH sequences With s1x—res1due
`their standard deviations are:
`_.——_———_~—_——
`
`(J
`(see Fig. 5(b)). The r.m.s. deviation of all N, C“,
`and O atoms is 0-96 A. The McP0603 H2 loop is
`shown as conformation 4 in Figure 3. The sequences
`in these regions are:
`
`
`
`
`
`
`52b 52c 53 54 5552a 71
`
`Tyr Arg
`Lys
`Asn
`Lys Gly
`Asn
`McPCfiOB
`—————_________————~
`4-4-20
`Asn
`Lys
`Pro
`Tyr
`Asn
`Tyr
`Al‘g
`
`$1
`4’1
`Angle
`11/4
`(#4
`Ill;
`(#3
`1P2
`4’2
`-18
`— 7S
`22
`65
`77
`—95
`—35
`—61
`Mean (deg)
`12
`13
`ll
`11
`14
`12
`8
`12
`Standard deviation (deg)
`N
`
`()f the nine loops in Table 2, excluding J539, seven
`have a Gly in the third position, like K011, 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 HyHlCL-S and J539 have Gly
`in the fourth position of the loop:
`
`the residues found at this position are
`H2 loops,
`Gly, Asn or Asp (Kabat et at., 1987). It is interesting
`to note in this context that the Lys at position 54 m
`McP0603 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 VH domain shows that the
`determinants of the conformations of four—residue
`H2 loops are not entirely within the sequence of the
`loop itself, but
`involve the packing of the loop
`against the rest of the structure.
`In Flgures 4 and 5 we show, for pairs of anti-
`bodies of known structure, the relative positions of
`the H1 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
`all the known structures. But the positions of the
`H2 loops are 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
`
`
`
`53 5452a 55M
`
`
`
`
`
`Ser
`Gly
`Asp
`Asp
`KUL
`11y
`Ser
`Asp
`Pro
`.1539
`
`H y H 1‘] L-5 Gly Pro (I ly Ser
`
`
`
`
`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
`KUL (see Fig 4(b) and Fig. 5(a)). The r.m.s, devia—
`tion in the position of the H2 main-chain atoms in
`11539 and HyHEL-5 is 1-9 A; for J539 and KOL it is
`03 A. The residues of H2 in J539 make no non-
`bonded contacts to residues other than those in H1
`and Arg7l and Asn73 (see Fig. 2).
`
`(c) Six—residue [12 regions
`
`the H2 loops are six-
`In McP(3603 and 4-4-20,
`residue hairpins. Their conformations are similar
`
`

`

`Position and Conformation of the H2 Loop
`
`179
`
`
`
`
`‘\,
`
`ArgTI/Val 7|
`
`Phe 29/Ile 29
`
`(0)
`
`
`
`Figure 4. The relative positions of the H1 and H2 hypervariable regions and of framework residue 71, in different pairs
`of'immunoglObulins. The H1 and H2 regions are represented by their C“ atoms. The pos1tions shown here are those found
`after the superposition of the VH framework residues (see text). (a) NEWM (00115111110115 111135) and HyHEL~lO (broken
`lines).
`(13) KOL (continuous lines) and J539 (broken lines).
`
`side-chains of these arginine residues are buried.
`They fOI‘m hydrogen bonds to main-chain atoms of
`residues in the H1 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 71 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 pack 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-S, the Pr052a
`side—chain would occupy the same space as the side-
`chain of Arg71 (Fig. 5(a)). The set of torsion angles
`
`that move the side-chain of Pro52a away 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 Arg7l
`has its side-chain buried, and is hydrogen bonded to
`the main—chain of H1 and H2, as in KOL and J539
`(Fig. 5(b)). The Tyr at the sixth position (55) packs
`against Arg7l.
`
`5. The Role of Residue 71
`
`These observations can be summarized as follows.
`(1) 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 H1 and H2
`loops close together and puts four-residue H2 loops
`in conformation 2 (Fig. 3).
`(2) Residue 7] is an arginine. The side-chain of
`
`

`

`
`
`180 A. Tmmontano et a1.
`
`Pro 520
`
`
`
`Figure 5. The relative positions of
`in pairs of
`the HI and H2 hypervariable regions and of framework residue 71,
`known 1mmunoglohulin structures. Th
`c H] and HQ regions are represented by their (3“ atoms. The positions shown here
`are those found after the supe
`rposition of the VH framework residues (see text). (a) HyHEL-5 (continuous lines) and
`J53”) (“Wk“) lines); (1)) Mcl’(7603 (continuous lilies) and 4-4-20 (broken lines)
`
`the argininc is buried between H] and H2, and
`forms hydrogen bonds with the main-chain in both
`loops. The H2 loop is displaced from H] with
`residue 52a on the surface. Four—residue H2 loops
`have conformation 3 (Fig. 3).
`ln Fab NCd-l residue 71 is a Leu, intermediate in
`size.
`In
`the
`structure
`of
`the
`Fab N041—
`neuraminidase
`complex
`( lolman
`at
`(1L,
`1987;
`Chothia (at (LL, 1989), H2 has the HyHEL—5 confor—
`mation. Residue 52a in N04] is a Thr, smaller than
`the Pro at the corresponding position in HyHEL—S;
`as a result the shift in Hz produced by the Leu is
`reduced.
`
`For six-residue H2 loops we have information for
`Mcl’C603 and 4-4-20, in which residue 71 is Arg. All
`known V” sequences that contain six-residue H2
`loops have Arg at position 7] (see below).
`
`6. Applications to Structure Prediction. The H2
`Regions in Immunoglobulins 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
`et al.
`(1987) have
`collected the known immunoglobulin sequences. We
`found in this collection 302 VH 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.
`
`

`

`
`Position and Conformation of the H2 Loop
`181
`
`This implies that they have conformations similar
`to that of H2 in NEWM and HyHEL-IO: an impli—
`cation supported by the prediction of the conforma—
`tion of the H2 region in D13 (Chothia et (11., 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 71 and Gly, Asn or Asp at position 54. For
`these we have the clear expectation that the H2
`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 Asp7 at position 55; and Val,
`Lee 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
`HyHlC L-5.
`Most of the 41 other four-resi(.lue 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 HyHlCli-5, depending on
`the residue at position 71,
`is more tentative. The
`structure of Fab NQlO has recently been deter—
`mined (Spinelli at al., unpublished results). In NQIU,
`the sequence of H2 is S-G-S-S, with Arg in position
`71 (Berek ct (1.1., 1985). The occurrence of Ody at the
`second position of a four-residue hairpin is very
`unusual;
`it does not occur in any of the loops
`surveyed by Chothia & liesk (1987) and Sibanda (4t
`ai. (1989). (KOL has Gly at the third position of the
`loop; and HyHlCL-S has Gly at the fourth position
`of the loop). The conformation and position of H2 in
`NQIO are the same as in KOL: The r.m.s. deviation
`of all N, C“, c and 0 atoms of H2 is 03%; the
`r.m.s. deviation of all N. C“, C and 0 atoms 0le
`and H2 together is 0-43 A (Chothia et (11.. 1989), 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 (11y, Asn or Asp at 54. The conser—
`vation at these sites suggests these HZ regions have
`conformations close to that in lVlcP(‘/6()3 and 4-4—20.
`
`7. Applications to Antibody Engineering
`
`The ability to transplant l‘1ypervariabli-2 regions of
`non-human origin to human frameworks
`is of
`medical
`importance (Reiclnnani‘i ct
`(LL, 1988). 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-
`chain portions of close—packed segments of proteins
`can move relative to each other by l
`to 2 A, with
`little expenditure of energy ((Yhothia «I.
`(1.1.. 1983).
`and the apices of loops may well be able to move by
`larger amounts. Thus the effect on antigen binding
`of changing the conforn'iation. or the position and
`
`orientation, of a hypervariable 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.
`transplanted the antigen-
`(1986)
`Jones ct al.
`binding loops from the heavy chain of a' mouse
`antibody on to the framework ofa 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 ct (Ll.
`(1986) shows that both the mouse and human anti-
`bodies have Val 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.
`
`transferred the hyper-
`(1988)
`Verhoeyen at al.
`variablc regions of the heavy chain of the mouse
`ai'iti—lysozyn’ie antibody l)l.3 to the framework of
`the human antibody NICVVM. An affinity for lyso-
`zyme was
`retained, although reduced approxi-
`mately tenfold. Both 1)].3 and NEWM contain a
`(11y at position 55 of the heavy chain; at position 71
`l)l.3 contains liys and NICVVM contains Val. This
`would suggest that in the synthetic antibody H2
`has the correct confbri’i’iation but is displaced from
`the position in 1)] .3. 1n the 1)] .3—1ysozyme complex,
`the contacts made by H2 (residues 53 to 55) to the
`antigen involve residues (7}ly53 and Asp54 (Amit (at
`(LL, 1986). we cannot determine to what extent the
`slight loss of affinity by the synthetic antibody is
`associated with
`the molecular
`readjustments
`required to retain these contacts.
`Reichmann (at a]. (1988) reshaped an antibody by
`transplanting all six hypervariable regions from a
`rat antibody on to a human framework for both 1'11
`and VH domains. In this case H2 had six residues, as
`(loos MePCBO3. The parent rat antibody has Arg at
`position 71, but
`the human framework has Val.
`There is no known 17“ 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. VVl‘icther this is because
`the cavity created by the smaller residue does not
`significantly affect
`the conformation of the six—
`residue 1-12, 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 hypervariable region of VL (liesk & Chothia.
`1982; Chothia & liesk, 1987). In that case the nature
`of the framework residues is related directly to the
`class of
`the light chain: K or 1. The analysis
`presented here den‘ionstrates that a framework
`residue plays a major role in determining position
`
`

`

`
`
`182 A. Tramontano et a1.
`
`and conformation of a hypervariable region within
`one (class of domains, the V“.
`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
`)‘iattern. 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 ()ur previous analysis of
`the other hypervariablc regions (Chothia & Leek.
`1987),.Improve the accuracy of predicted immuno-
`glohulin structures.
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`Edited by A. filers/Lt
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