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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, U.S.A.
`
`Assistant Editor
`J. Karn
`MRC Laboratory of Molecular Biology
`Hills Road, Cambridge CB2 2QH, 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 IEW, U.K.
`M. Gottesman, Institute of Cancer Research, College of Physicians & Surgeons of Columbia University,
`701 W. 168th Street, New York, NY 10032, U.S.A.
`.
`P. von Hippel, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229, U.S.A.
`R. Huber, Max-Planck-Institut fiir Biochemie, 8033 Martinsried bei Miinchen, Germany.
`A. Klug, MRCLaboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K.
`
`Associate Editors
`C. R. Cantor, Human Genome Center, Donner Laboratory, Lawrence Berkeley Laboratory, University of California
`Berkeley, CA 94720, U.S.A.
`N.-H. Chua, The Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S.A.
`F. EF. Cohen, Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco,
`CA 94143-0446, U.S.A.
`D. J. DeRosier, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02254, U.S.A.
`W. A. Hendrickson, Department of Biochemistry & Molecular Biophysics, College of Physicians & Surgeons of
`Columbia University, 630 West 168th Street, New York, NY 10032, U.S.A.
`I.B. Holland,
`Institute de Genetique et Microbiologie, Batiment 409, Université de Paris XI, 91405 Orsay 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, U.S.A.
`V. Luzzati, Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, 91 Gif-sur-Yvette, 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, OR 97403-1229, U.S.A.
`J. H. Miller, Department of Microbiology, University of California, 405 Hilgard Avenue, Los Angeles, CA 90024, U.S.A.
`M. F. Moody, School of Pharmacy, University of London, 29/39 Brunswick Square, London WCIN 1AX, U.K.
`T. Richmond, Institut fiir Molekularbiologie und Biophysik, Kidgendssische Technische Hochschule, Honggerberg,
`CH 8093 Zurich, Switzerland.
`R. Schleif, Biology Department, Johns Hopkins University, Charles & 34th Streets, Baltimore, MD 21218, U.S.A.
`N. Es Sternberg, Central Research & Development Department, K.
`I. du Pont Nemours & Company, Wilmington,
`DE 19898, U.S.A.
`K. R. Yamamoto, Department of Biochemistry and Biophysics, School of Medicine, University of California,
`San Francisco, CA 94143-0448, U.S.A.
`M. Yanagida, Department of Biophysics, Faculty of Science, Kyoto University, Sakyo-Ku, Kyoto 606, Japan.
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`
`
`176 A. Tramontano etal.
`
`
`
`: Figure 1. Outline structure of the antigen-bindingsite.
`Thesite is formed by 6 loops of polypeptide (AW) linked
`to strands in fp-sheets (().
`eS
`
`Tn the immunoglobulins of known structure the
`conformations of the second hypervariable region in
`Vy (H2) differ. The position of the H2 with respect
`to the conserved framework is also variable. For
`example,
`in the Vy domains of immunoglobulins
`J539 and HyHEL-5, the H2 regions have the same
`num ber of residues. If the framework structures are
`superimposed, the C” atoms in residue 53, at the tip
`of H2, are found to differ
`in position by 63A
`(lA=0:1 nm). Here, we show that the variationsin
`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.
`
`Figure 2 shows the general structural context of
`H2 within the Vj domain.
`
`2. Co-ordinates and Calculations
`
`Protein structures used in this work are listed in
`Table 1. 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.
`Programswritten 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 Vy domains, the conserved f-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 Vy domains.
`
`3. The Conformations of H2 Loops
`In Vy sequences the second hypervariable region
`consists of a f-hairpin, comprising residues 50
`through 65 (Wu & Kabat, 1970; Kabat et al., 1987).
`In the known V,, 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 atomsofresidues 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 regionis illustrated in Figure 3:
`
`Figure 2. The structural context of H2 within the Vy domain of Fab 3539. H2 is shaded relatively darkly, H1 is
`shadedrelatively lightly. The thick brokencircle indicates the guanidinium group of Arg371.
`
`
`
`
`
`Position and Conformation of the H2 Loop 177
`
`Table 1
`Immunoglobulin heavy chain variable domains of known atomic structure
`
`
`Molecule
`H2 sequence
`Residue 71
`Reference
`
`
`NEWM
`HyHEL-10
`
`HyHEL-5
`KOL
`J539
`
`Y
`¥
`
`P
`D
`P
`
`H
`8
`
`G
`«G
`
`i
`§
`+
`D GS
`D
`§
`3
`
`Vv
`R
`
`A
`R
`R
`
`Saul et al. (1978)
`Padlanet al. (1989)
`
`Sheriffef al. (1987)
`Marquartet al. (1980)
`Suhet al. (1986)
`
`Satow et al. (1986)
`R
`Y
`K GN K
`N
`MePC603
`
`
`
`
`
`N &£& FY N YF R4-4-20 Herronet al. (1989)
`
`The H2 residues are those between positions 52 and 56(see text).
`
`o>0, W>0) (Sibandaet al., 1989). Both NEWM and
`HyHEL-10 havea glycine at this position:
`
`
`
` 53 54 55 71
`
`
`
`
`
`Val
`Gly
`His
`Tyr
`NEWM
`
`
`
`
`HyHEL-10 Arg Tyr Ser Gly
`
`andin both cases the Gly is in a ++ conformation.
`
`(b) Four-residue 12 regions
`The H2 loop of HyHEL-5 is a four-residue
`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 op
`conformation and the fourth in an a, 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 a, conformation and the first, second and fourth
`in the & conformation. This is shown as conforma-
`tion 3 in Figure3.
`
`the main-chain atoms of residues 56 to 60 form
`hydrogen bondsto those of residues 48 to 52 to form
`a B-hairpin. Sequence variations in these residues
`havelittle 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 knownstructures 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 NEWMand HyHEL-10,
`the H2 loop is a
`three-residue hairpin, residues 53 to 55. The NEWM
`H2 loopis shown as conformation | in Figure 3. The
`usual sequence requirement for this conformationis
`a Gly (or Asn or Asp) at the third position (residue
`55), which can take up a ++ conformation (thatis,
`
`DI-3
`NEWM
`HyHEL—1O
`
`HyHEL-5
`NC4I
`
`
`
`KOL
`J539
`NQIO
`
`McPC 603
`4—4-20
`
`Figure 3. The main-chain conformations of the 2nd hypervariable region in Vy domains in the immunoglobulins of
`known structure. The conformations are numbered| to 4. The immunoglobulins in which these conformations are found
`are listed under each number.
`
`
`
`
`
`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
`
`
`A
`Starting
`
`(A)
`Molecule (Protein Data Bank code)
`residue
`Sequence
`
`L
`G
`Q
`SS
`145
`Rhizopuspepsin (3APR)
`0-18
`A
`K
`I
`L
`10
`Subtilisin Carlsberg (28SEC)
`O19
`L
`N
`R
`Ss
`32
`Ribonuclease A (7RSA)
`0-22
`L
`+
`Q
`D
`142
`Pepsinogen (1 PSG)
`0-22
`K
`G
`N
`E
`35
`434 repressor protein (1 R69)
`0:22
`D GN
`A
`57
`Calmodulin (3CLN)
`023
`D
`@G@
`D
`K
`21
`Calmodulin (3CLN)
`O24
`R
`x
`lil
`K
`166
`Adenylate kinase (3ADK)
`0-28
`D
`S$
`G
`P
`353
`Fab J539
`0-29
`
`
`
`
`
`Cytochrome ¢551 (451C) 9 N K G@0:29 C
`
`
`A, root-mean-square deviation of N, C*, C and O atomsof residues 53 to 56 of the Vy domain of KOL
`andwell-fitting regions from other known structures.
`
`(see Fig. 5(b)). The r.m.s. deviation of all N, C*, C
`and O atomsis 0-96 A. The McPC603 H2 loop is
`shown as conformation 4 in Figure 3. The sequences
`in these regions are:
`
`
`
`
`
`
`52b 2c 53 54 5552a 71
`
`Tyr Arg
`Lys
`Asn
`Lys Gly
`Asn
`McPC603
`Tyr Arg
`Asn
`Tyr
`Lys
`Pro
`Asn
`4-4-20
`eng
`
`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. 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 andthe nine best-fitting non-homologous loops,
`In both structures residue 54 is in the a confor.
`the average valuesof the conformational angles and
`mation. In the other Vy sequences with six-residue
`their standard deviations are:
`—_—_eeeeeeeeSeSFSFSMmmsshseseFSFSSSSSSSSSMSSes
`Angle
`Py
`Vy
`Va
`$4
`ws
`3
`Wo
`b2
`—18
`=78
`22
`65
`77
`—95
`Mean(deg.)
`—61
`—35
`12
`13
`ll
`ll
`14
`12
`Standard deviation (deg.)
`12
`8
`—_—_eeeeeeeeeeeeESSSFSmFmmssseheFeseseseseee
`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.
`deviationless than 0-5 A, noneis 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:
`
`the residues found at this position are
`H2 loops,
`Gly, Asn or Asp (Kabatet al., 1987). It is interesting
`to note in this context that the Lys at position 54 in
`McPC603is 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 Vy domain shows that the
`determinants of the conformations of four-residue
`H2loopsare 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 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 H1 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 55ee
`
`
`
`
`
`Ser
`Gly
`Asp
`Asp
`KOL
`aly
`Ser
`Asp
`Pro
`J539
`
`
`
`
`Pro Gly SerHy HEL-5 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 atoms in
`J539 and HyHEL-5is 1-9 A; for J539 and KOLitis
`0-3 A. The residues of H2 in J539 make no non-
`bonded contacts to residues other than those in H1
`and Arg71 and Asn73 (see Fig. 2).
`
`(c) Sia-residue [12 regions
`In MePC603 and 4-4-20,
`the H2 loops are six-
`residue hairpins. Their conformations are similar
`
`
`
`»
`
`Phe 29/Ile 29
`
`\,
`
`Position and Conformation of the H2 Loop
`
`179
`
`
`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 H2regions are represented by their C* atoms. The positions shownhere are those found
`after the superposition of the V,, 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 arginine residues are buried.
`They form hydrogen bonds to main-chain atoms of
`residues in the H] 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
`whichresidue 71 is an Arg, residue Prod2a in the H2
`loop is on the surface.
`In HyHEL-5,
`in which
`residue 71 is an Ala, Prod2a 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 Vy
`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 Prod2a
`side-chain would occupy the same spaceas 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 Arg71
`hasits side-chain buried, and is hydrogen bonded to
`the main-chain of H] 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 asfollows.
`(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 HI and H2
`loops close together and puts four-residue H2 loops
`in conformation 2 (Fig. 3).
`(2) Residue 71 is an arginine. The side-chain of
`
`
`
`
`
`180 A. Tramontano etal.
`
`
`
`Pro 52a
`
`Figure 5. The relative positions of
`in pairs of
`the Hl and H2 hypervariable regions and of framework residue 71,
`known immunoglobulin structures. Th
`e H1 and H2 regions are represented by their C* atoms. The positions shown here
`are those foundafter the supe
`rposition of the Vy framework residues (see text). (a) HyHEL-5 (continuous lines) and
`J539 (broken lines); (b)
`MePC603 (continuous lines) and 4-4-20 (broken lines)
`
`the arginine is buried between H1 and H2, and
`forms hydrogen bonds with the main-chain in both
`loops. The H2 loop is displaced from H1 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-
`neuraminidase
`complex
`(Colman
`et
`al.,
`1987:
`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 12 produced by the Leuis
`reduced,
`Forsix-residue H2 loops we have information for
`McPC603 and 4-4-20, in which residue 71 is Arg. All
`known Vy sequences that contain six-residue H2
`loops have Arg at position 71 (see below).
`
`6. Applications to Structure Prediction. The H2
`Regions in Immunoglobulins of
`UnknownStructure
`
`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 Vy domains of
`unknown structure. Kabat
`et al.
`(1987) have
`collected the known immunoglobulin sequences. We
`found in this collection 302 Vy sequences for which
`all, or almost all, residues in the region 50 to 75 are
`known. Ofthese 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-10: an impli-
`cation supported by the prediction of the conforma-
`tion of the H2 region in D1.3 (Chothia et al., 1986).
`At position 71, Arg or Lys occurs in 43 sequences
`and Val or Leuin 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 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.
`Mostof the 41 other four-residue H2 regions do
`not have a Gly, Asn or Aspat 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 NQUO,
`the sequence of H2 is S-G-S-S, with Arg in position
`71 (Berek e¢ 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 Glyatthe third position of the
`loop; and HyHEL-5 has Gly at the fourth position
`of the loop). The conformation andposition of H2 in
`NQ10 are the same as in KOL: The r.m.s. deviation
`of all N, C*, © and O atoms of H2 is 0°39 A; the
`r.m.s. deviation of all N, C%, C and O atoms of H1
`and H2 togetheris 0-43 A (Chothia e¢ al., 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 Tyrat position 55, Arg at 71 and
`all but two have Gly, Asn or Aspat 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 (Reichmannef al., 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 sameresidues at thosesites
`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 moverelative to each other by 1
`to 2 A, with
`little expenditure of energy (Chothia ¢¢ al., 1983),
`andthe apices ofloops 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 hypervariable region will depend
`upon whetherthe region is involved in binding and,
`if it is, on how much energyis 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 ef al.
`binding loops from the heavy chain of a mouse
`antibodyon to the frameworkof a humanone. They
`observed that the synthetic product, when boundto
`the original mouselight chain, had the same affinity
`for
`the hapten as
`the original mouse antibody.
`Inspection of the two sequences used by Jonesetal.
`(1986) shows that both the mouse and humananti-
`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.
`Verhoeyen et al.
`(1988)
`transferred the hyper-
`variable regions of the heavy chain of the mouse
`anti-lysozyme antibody D1.3 to the framework of
`the human antibody NEWM. Anaffinity for lyso-
`zyme was
`retained, although reduced approxi-
`mately tenfold. Both D1.3 and NEWMcontain a
`Glyat position 55 of the heavychain; at position 71
`D1.3 contains Lys and NEWMcontains 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 D1.3-lysozymecomplex,
`the contacts made 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 antibodyis
`associated with
`the molecular
`readjustments
`required to retain these contacts.
`Reichmannef al. (1988) reshaped an antibody by
`transplanting all six hypervariable regions from a
`rat antibody on to a human framework for both Vj,
`and Vj 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 Vy sequence with the combina-
`tion a six-residue H2 and Val at position 71
`(sce
`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 hypervariable region of Vj (Lesk & Chothia,
`1982: Chothia & Lesk, 1987). In that case the nature
`of the frameworkresidues is related directly to the
`class of
`the light chain: x or 4. The analysis
`presented here demonstrates that a framework
`residue plays a major role in determining position
`
`
`
`
`
`182 A. Tramontanoet al.
`
`(1982). J. Mol. Biol. 160,
`
`and conformation of a hypervariable region within
`one class of domains, the Vy.
`Wehavealso 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 ofits 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 involvedin 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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