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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
`
`1’. Chambon, Laboratoire de Génétique lVloléculaire des Eucaryotes du CNRS, Institut de Chimie Biologique.
`Faculté de Médecine, 11 Rue Hum-ann, 67085 Strasbourg Cedex, France.
`A. R. Fersht, University Chemical Laboratory, Cambridge University, Lensfield Road, Cambridge CB2 IEW, U.K.
`M. Gottesmann, Institute of Cancer Research, College of Physicians & Surgeons of Columbia University,
`701 W. 168th Street, New York, NY 10032. U.S.A.
`P. van Hippel, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229, U.S.A.
`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,1(_
`
`Associate Editors
`
`0. R. Cantor, Human Genome Center, Donner Laboratory, Lawrence Berkeley Laboratory, University of Ca]if01-ma‘
`Berkeley, CA 94720, U.S.A.
`‘
`N.-H. (Jhua, The Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S_A_
`I". E. Cohen, Department of Pliarmaceutical Chemistry, School of Pharmacy, University of California, San l*‘rancisco,
`CA 94143-0446, U.S.A.
`I). J. I)cl€()sier, Rosenstiel Basic Medical Sciences Research Center. Brandeis University, Waltham, MA 02254. U.S.A.
`W. A.
`I-Ie'rLdr‘lclcson, Department of Biochemistry & Nlolecular Biophysics. College of Physicians & Surgeons of
`Columbia University, 630 West 168th Street, New York. NY l0032, U.S.A.
`[.13. Holland,
`Institute de Genetique et Microbiologie’ Bétiment 409, Université de Paris XI. 9140.3 Orsay Cedex 05,
`France.
`‘
`B. Honig, Department of Biochemistry & Molecular Biophysics, College of Pliysicians & 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 (lif'—sur—Yvet,t.(-., Fmm-e,
`J. L. Mandel, Laboratoire de Génétique lVIole'culaire 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 W(‘/1N lAX, UK,
`T. Riclmcond, Institut fiir Molekularbiologie und Biophysik, Eidgeniissische Technische Hochschule. Ho'nggerberg,
`CH 8093 Zurich, Switzerland.
`R. Schleif, Biology Department, Johns Hopkins University. Charles & 34th Streets, Baltimore, MD 21218, U.S_A_
`N. L. Stcrnberg, Central Research & Development Department, E.
`I. du Pont Neinours & Company, Vllilmington,
`DE 19898, U.S.A.
`K. R. Yamamoto, Department of Biochemistry and Biophysics, School of Medicine, University of California.
`San Francisco, (VA 94143-0448, U.S.A.
`'
`M. Yanaglda, Department of Biophysics, Faculty of Science, Kyoto University, Sakyo-Kn, Kyoto 606, .Japan.
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`

`
`
`
`1'7 6 A. Tiramontano et al.
`
`
`
`‘ Flg_ure_1..()utline stru<-.ture of the antigen-binding site.
`'l he site is formed by 6 loops of polypeptide (/W) linked
`to straiids in /3-slieets ([1),
`j5
`
`hi the iminui’ioglol)ulins of known structure the
`conformations of the second liypervariable region in
`VH (H2) difier. The position of the H2 with respect
`to the conserved framework is also variable. For
`‘”:*_’”"lJl‘3.,
`in the VH domains of immunoglobulins
`-1.039 and HyHl4lL-5, the H2 regions have the same
`nu m bei‘ of residues. If the framework structures are
`supei'iin1)()se(l, the C” atoms in residue 53, at the tip
`(Eli
`(‘H26 are found to difl°er
`in position by 6'3 A
`mm’) *1 l_ulC{‘l'lYI‘"l -I
`sliowc that the variationsm
`mM_(;[-.nm,Lii(":L )11le.S])(()ii H2, its position and
`‘MW pa” 0", are cotlpr.
`, and that they flehend in
`:5
`lie natuie of the amino acid residue
`that occupies position 71
`in
`the
`l‘ieavy—chain
`framework.
`
`Figure 2 shows the general structural context of
`H2 within the V“ 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 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.
`(1987). In VH domains, the conserved ,6-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 V” domains.
`
`3. The Conformations of H2 Loops
`
`In VH sequences the second hypervariable region
`consists of a [3-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 2. The structural context of H2 within the V“ domain of Feb J539. H2 is shaded relatively darkly, H] is
`slizuled relatively lightly. The thick broken circle indicates the guanidinium group of Arg37l.
`
`
`
`

`
`
`
`Pos'itz'on and Conformation of the H2 Loop 177
`
`Table 1
`
`Immunoglobul in he(wg/ cI2,(Lin vavriable dorrrmins of /cnown ato/rmfc structure
`
`
`
`Molecule
`
`H2 sequence
`
`Residue 71
`
`Reference
`
`NEWM
`HyHEL.1()
`
`HyH EL-5
`K0],
`J539
`
`Y
`Y
`
`l’
`])
`1)
`
`H
`S
`
`(l
`I)
`1)
`
`G
`(_;
`
`S
`(,‘.
`S
`
`I
`S
`(1
`
`V
`R,
`
`A
`K
`R
`
`Saul at (1.1. (1978)
`Padlan of al. ( I989)
`
`Sheriff cl ((1. (1987)
`Marquart 9! (Li. (1980)
`Suh cl at. (1986)
`
`Satow at 121‘ (1986)
`R
`Y
`K
`N
`G
`K
`N
`M(-.P(,‘6()3
`
`
`
`
`
`
`
`
`N K P Y N Y R4-4-20 Herron at al. (1989)
`
`The H2 residues are those between positions 52 and 56 (see text).
`
`the main—ch-ain atoms of residues
`
`to 60 f0I'm
`
`hydrogen bonds to those ofresidues 48 to 52 to form
`a [3-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
`or 53 to 55, we refer to
`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,
`i968; Efimov, 1986; Sibanda & Thornton, 1985;
`Sibanda at 01.,
`I989). Particular‘ conformations are
`usually associated with characteristic sequence
`patterns. The positions of Gly, Asn, Asp and PTO
`residues are important because these residues allow
`main-chain conformations that
`in other residues
`cause strain.
`
`(a) 7’}m‘ee—7'es~idue [12 regions
`
`the H2 loop is it
`In NEWM. and HyI-IEL-I0,
`three-residue hairpin, residues 53 to 55. The NEWM
`H2 loop is shown as conformation l 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 (tl‘1a1> i-S‘,
`
`gb>0, zp>0) (Sibanda ct (LL, 1989). Both NEWM and
`Hy.Hl§lL—l() have a glycine at this position:
`
`
`
`
` 53 54 7|
`
`
`
`Val
`Gly
`His
`Tyr
`NEWM
`
`l'lyHl*lL— l O Arg T_yr Sci" (lly
`
`
`
`
`and in both cases the Gly is in a + + conformation.
`
`(b) Four-2‘es'2Id'ue I72 regifons
`
`The H2 loop of HyHEL-5 is a four-residue
`hairpin, residues 52a to
`This is shown
`confor-
`mation 2 in Figure 3. The conformation is close to
`the one most commonly observed in four—rcsiduc
`turns, in which the first three residues are in an oak
`conformation and the fourth in an o¢L conformation.
`These turns normally require Gly in the fourth
`position (Efimov, 1986; Sibanda & Thornton, 1985;
`Sibanda at (Ll., 1989), as observed in HyHEL-5.
`The H2 regions in KO L and J539 form four«
`residue turns with a conformation different from
`
`Hyl-{EL-5. They both have the third residue (54) in
`the (XL conformation and the first, second and fourth
`in the ozk conformation. This is shown as conforma-
`tion 3 in Figure 3.
`
`
`
`Di-3
`NEWM
`HyHEl_~lO
`
`HyHEL—5
`NC4|
`
`KOL
`J539
`NOIO
`
`MCPC 603
`4 — 4 — 20
`
`Figure 3. The main-chain conformations of the 2nd hypervariable region in V“ domains in the immunoglobulins of
`known structure. The conformations are numbered 1 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—cha'in cortformattons the same as that of the H2
`loop of K0L
`
`
`
`A
`(A)
`
`Molecule (Protein Data Bank code)
`
`Starting
`residue
`
`Sequence
`
`L
`G
`Q
`S
`145
`Rliiwpuspepsin (3APR)
`0-18
`A
`K
`I
`L
`10
`Subtilisin Uarlsberg (ZSEC)
`0'19
`L
`N
`R
`S
`32
`ltibonuclease A (7RSA)
`0'22
`L
`G
`Q
`D
`142
`Pepsinogen (IPSG)
`022
`K
`G
`N
`E
`35
`434 repxessor protein (lR69)
`0'22
`N
`G
`D
`A
`57
`(lalmodulin (3(}LN)
`0'23
`D
`G
`D
`K
`21
`(falmodulin (3(3LN)
`024
`I
`G
`R
`K
`166
`Adenylate kinase (3AI)K)
`0-28
`G
`S
`D
`P
`353
`Fab J 539
`029
`
`
`
`
`
`
`Cytochrome 0551 (4510) 9 N K G(1-29 (3
`
`A, root-mean-square deviation of N, C‘, (1 and () 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 (il 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 oc,_ conform
`the average values of the conformational angles and
`mation. In the other VH sequences with six-residue
`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 McPC6()3 H2 loop is
`shown as conformation 4 in Figure 3. The sequences
`in these regions are:
`
`
`
`
`
`
`52b 52c 53 5-1 5552a 71
`
`Tyr Are
`Lys
`Asn
`Lys Gly
`Asn
`Mcr(16o3
`
`4-4-20 A"g Asn Lys Pro Tyr Asn Tyr
`
`
`
`
`
`
`
`3,01
`div,
`Angle
`l//4
`«#4
`Ill;
`do
`W2
`¢z
`— 18
`— 78
`22
`65
`77
`-95
`-35
`-61
`Mean (deg)
`12
`13
`ll
`11
`1-1
`12
`8
`12
`Standard deviation (deg.)
`
`
`excluding J539, seven
`()f the nine loops in Table
`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 HyHl<]Ii-5 and J539 have Gly
`in the fourth position of the loop:
`
`55
`54
`53
`52a
`___j
`
`Ser
`Gly
`Asp
`Asp
`l\'()l.
`Gly
`Ser
`Asp
`Pro
`«I539
`
`H y H E L-5 U-ly Pro (1 ly Sex‘
`
`
`
`
`The position of Gly in J539 should imply a confor-
`mation of H2 similar to that of HyHI43l.-5. Instead
`the conformation observed in J539 is the same as in
`KO]. (see Fig. 4(b) and Fig. 5(a)). The r.m.s. devia-
`tion in the position of the H2 rnain-chain atoms in
`J539 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.
`
`(c) S'ix—resid'ac 1],? regions
`
`the H2 loops are six-
`In McP(36()3 and 4-4-20,
`residue hairpins. Their conformations are similar
`
`the residues found at this position are
`H2 loops,
`Gly, Asn or Asp (Kabat et at., I987). It is interesting
`to note in this context that the Lys. at position 54 In
`McPC6()3 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 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 7l.
`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
`
`

`
`Position and Oonformatwn of the H2 Loop
`
`17 9
`
`
`
`
`“.
`
`Phe 29/Ila 29
`
`Arg 7|/VCII 7|
`
`
`
`Figure 4. The relative positions of the H1 and H2 hypervariable regipiis and of framework residue 71, in different pairs
`ofimmunoglobulins. The H1 and H2 regions are represented by their
`‘atoms. The posltions shown here are those found
`after the superposition of the V“ framework residues (see text). (a) IVEWM (00m9mU0"S lmesl and HYH E1440 ibmkel‘
`lines). (b) KOL (continuous lines) and J539 (broken lines)-
`
`side-chains of these arginine residues are buried.
`They f0I‘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-5, the Pro52a
`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 I-’ro52a 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. ’I’mm0'nia'rLo et al.
`
`Pro 520
`
`Figure 5. The relative positions of
`in pairs of
`the HI and H2 hypervariable regions and of fra.mework residue 71,
`known immunoglohulin structures.
`The 1-1] and H2 regions are represented by their C“ atoms. The positions shown here
`are tliose found afte
`1- the superposition of the VH framework residues (see text). (a) HyHEL-5 (continuous lines) and
`(1)) Mcl’(3603 (rontinuous lines) and 4-4-20 (broken lines)
`
`J539 (l"‘0l<Ull H1168):
`
`the argininc is buried bctwccn H1 and H2, and
`forms Iiydrogen bonds with the main-chain in both
`loops. The H2 loop is displaced from H] with
`rosiduc 52a on the surface. Four—residue H2 loops
`have conformation 3 (Fig. 3).
`ln Fab NC4-1 residue 71 is a Leu, intermediate in
`size.
`In
`the
`structure
`of
`the
`Fab N041-
`neuraminidase
`complex
`( Iolman
`at
`al.,
`1987;
`(Ihothia at al., 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-5;
`as a result the shift in 1112 produced by the Leu is
`reduced.
`
`For six—rcsidue H2 loops we have information for
`Mc1’C6()3 and 4-4-20, in which residue 71 is Arg. All
`known V“ 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
`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 V“ domains of
`unknown structure. Kabat
`et al.
`(1987) have
`collected the known immunoglobulin sequences. VVe
`found in this collection 302 V“ 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
`
`

`
`Posit'ion and (]on_formati(m of the H2 Loop
`
`181
`
`This implies that they have conformations similar
`to that of H2 in NEWM and HyHl7.L-10: an impli-
`cation supported by the prediction of the conforma-
`tion of the H2 region in Dl.3 (Chothia ct 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 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 K()L and J539.
`Another 99 sequences have Pro at position 52a; Gly,
`or in a few cases Asn or Asp, at position 55; and Val,
`lieu or Ala at 7]. 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
`
`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 HyHlCL-5, depending on
`the residue at position 71,
`is more tentative. The
`structure of Fab NQIO has recently been deter-
`mined (Spinelli at al., unpublished results). In NQIO.
`the sequence of H2 is S-G-S-S, with Arg in position
`7] (Berek ct (LL, 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 & liesk (l987) and Sibanda at
`(LL (I989). (KOL has Gly at the third position of the
`loop; and HyHlCL-5 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 O atoms of H2 is 0'39A; the
`r.m.s. deviation of all N.
`I“, C and 0 atoms of H1
`and H2 together is 0'43 A (Chothia et al., 1989). This
`then confirms the importance of the residue at
`position 7] 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 7] and
`all but two have (lly, Asn or Asp at 54. The conser-
`vation at these sites suggests these H2 regions have
`conformations close to that in l\’lcl’(‘/(’503 and 4-4-20-
`
`7. Applications to Antibody Engineering
`
`The ability to transplant hypervari-able 1'0?-’§l""S "i:
`non-human origin to human frameworks
`is of
`medical
`importance (lieiclmiaim ct (lf.,
`l988). 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 <-lose—packed segments of proteins
`can move relative to each other by l
`to 2 A, with
`little expenditure of energy (Uhothiet N 01.. 1953),
`and the apices 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 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 er! 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.
`(l98()') shows that both the mouse and human anti-
`bodies have Val at position 7], and therefore we
`should expect
`the four-residue H2 loop from the
`mouse antibody to retain its <-.onformation and posi-
`tion on transfer to the human framework.
`
`transferred the hyper-
`(1988)
`(L1.
`Verhoeycn et
`Variable regions of the heavy chain of the mouse
`anti—|ysozyme antibody l)l.3 to the framework of
`the human antibody NICVVM. An afiinity for lyso—
`zyme was
`retained, although reduced approxi-
`mately tenfold. Both D133 and NTCWM contain a
`Gly at position
`of the heavy chain; at position 7l
`l)l.3 contains Lys and NICVVM contains Val. This
`would suggest that in the synthetic antibody H2
`has the correct eonformation but is displaced from
`the position in l)l .3. In the D1 .3—lysozyme complex,
`the contacts made by H2 (residues 53 to 55) to the
`antigen involve residues (,}ly5.‘—3 and Asp54 (Amit ct
`(LL, I986). VVe cannot determine to what extent the
`slight loss of afiinity by the synthetic antibody is
`associated with
`the molecular
`rcadjustn'1ents
`required to retain these contacts.
`Reichmann ct (1.1. U988) reshaped an antibody by
`transplanting all six hypervariable regions from a
`rat antibody on to a human framework for both 1",‘
`and V“ domains. In this case H2 had six residues, as
`does McP(J6()3. The parent rat antibody has Arg at
`position 7], but
`the human framework has Val.
`There is no known V“ sequence with the combina-
`tion a six—residuc H2 and Val at position 7]
`(see
`above). The synthetic antibody has an afiinity close
`to that of the rat original. VVhcther this is because
`the cavity (:reate('l by the smaller residue does not
`significantly aflect
`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 VL (liesk & (lhothia,
`1982; Chothia & Lesk, I987). ln 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 demonstrates that a framework
`residue plays a major role in determining position
`
`

`
`182
`
`A. Tmmontano et al.
`
`and conformation of a hypervariable region wz'thz'n
`one rtlass 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
`<:on1'ormation 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
`1l1(:()I‘I‘(\.(‘L.
`
`The results reported here will help in under-
`stai'1(1iz1g the molecular mechanisms involved in the
`generation of antibody diversity, extend the rules
`governing scqm-.nce—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-
`glohuhn structures.
`
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