25. Oiiier, C. D. Tectonics and Landforms (Longman. New York. 1981).
`
`Petrol. 21, 629-650 (1980).
`
`32. King. L C. South African Scenery 2nd edn (Oliver and Boyd, Edinburgh, 1951)
`33. King, L. C. The Morphology of the Earth (Oliver and Boyd, Edinburgh. 1962)
`34. Partndge, T. C. & Maud, R R S Afr. geol. J 90, 179-208 (19871.
`35. Cahen. L., Snelling, N. J., De!hal, J. & Vail, J. R. The Geochronology and Evolution of Africa (Clarendon,
`
`ARTICLES
`
`31. Craddock, C. (ed.) Antarctic Geoscience (University of Wisconsin Press. Madison, 1982)
`
`O'ford. 1984).
`36. Brown. R. W. 6th Int. Conf. Fission Track Dating Abstr. Vol. Universite de Franche-Comte. Besan.;on.
`1988)
`
`26. Fitch. F. J & Miller. J A. Spec. Pubis geol. Soc S Afr. 13, 247-266 (1984).
`27. co,, K. G. J
`28. England, P. C. & Molnar. P. Geology (in the press).
`29. Petri. S. & Fulfar6, V. J. Geologia do Brasil (Editora da Universidade de Sao Paulo. 1983)
`30. Tankard. A. J. et al. Crustal Evolution of Southern Africa (Springer, New York. 1982)
`Conformations of immunoglobulin
`hypervariable regions
`
`Cyrus Chothia*t, Arthur M. Lesk**, Anna Tramontano*, Michael Levitf§,
`
`
`
`
`
`A. Padlan#, Sandra J. Smith-Gillll, Gillian Air11, Steven Sheriff#**, Eduardo
`
`
`David Davies#, William R. Tuliptt, Peter M. Colmantt, Silvia SpinelliH,
`Pedro M. AlzariH & Roberto J. Poljak**
`* MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
`t European Molecular Biology Laboratory, Meyerhofstrasse 1, Postfach 1022.09, D-6900 Heidelberg, FRG
`t Christopher Ingold Laboratory. University College London, 20 Gordon Street. London WC1H OAJ, UK
`§ Department of Cell Biology, Stanford University Medical School, Stanford, California 94305, USA
`tt CSIRO Division of Biotechnology, 343 Royal Parade, Parkville 3052, Australia
`H Unite d'lmmunologie Structurale, Departement d'lmmunologie, lnstitut Pasteur, 25 rue du Dr Roux, 75724 Paris, France
`
`II National Institute of Cancer and # National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,
`
`Maryland 20892, USA
`�Department of Microbiology, University of Alabama, Birmingham, Alabama 35294, USA
`
`Kabat et al. 5 found conserved
`
`Paz et a!.7, showed that some of the hypervariable
`
`(Fig. la). Within the
`
`1 •2• The specificity
`
`regions 3•4•
`
`angles ¢, 1/1 or w, are primarily
`
`Table I). Examination
`
`at these sites (Fig. l and
`
`model, antibodies have only a few main-chain conformations
`
`
`On the basis of comparative studies of known
`
`
`
`
`
`or 'canonical structures' for each hypervariable region. Most
`
`antibody structures and sequences it has been
`
`
`
`sequence variations would only modify the surface provided by
`
`argued that there is a small repertoire of main­
`
`
`
`the side chains on a canonical main-chain structure. Sequence
`
`
`
`changes at a few specific sets of positions would switch the main
`
`chain conformations for at least five of the six
`
`
`chain to a different canonical conformation.
`
`
`hypervariable regions of antibodies. and that the
`
`
`particular conformation adopted is determined by
`Canonical structure model
`a few key conserved residues. These hypotheses
`Experimental evidence indicates that the canonical structure
`
`
`
`
`
`
`
`model describes the relationship between amino-acid sequence
`
`are now supported by reasonably successful pre­
`
`and structure for at least five of the six hypervariable regions5-9.
`
`
`dictions of the structures of most hypervariable
`
`residues at sites within certain
`
`regions of various antibodies, as revealed by
`
`
`sets of hypervariable regions and suggested that they had a
`
`
`
`structural role. Padlan and Davies6, and more recently de la
`
`comparison with their subsequently determined
`regions
`structures.
`
`in the immunoglobulins of known structure have the same
`
`
`
`main-chain conformation in spite of several differences in
`of THE relationships between the amino-acid sequences
`
`
`sequence.
`
`
`immunoglobulins and the structures of their antigen-binding
`Chothia and Lesk8 identified the residues that through pack­
`
`
`
`
`
`sites are important for understanding the molecular mechanisms
`
`
`
`ing, hydrogen bonding, or the ability to assume unusual values
`
`
`
`of the generation and maturation of the immune response and
`
`responsible for
`of the torsion
`
`
`
`
`for designing engineered antibodies. Antigen-binding sites are
`
`
`
`regions in the main-chain conformations of the hypervariable
`
`formed by six loops of polypeptide, the hypervariable regions;
`
`
`the structures then known-the Fab fragments of NEW (ref.
`
`three from the variable domain of the light chain (VL) and three
`10), McPC603 (ref. II), KOL (ref. 12) and J539 (ref. 13) and
`
`
`from the variable domain of the heavy chain (VH), denoted Ll,
`the VL domains of REI (ref. 14) and RHE (ref. 15). The
`L2, L3, and HI, H2, H3, respectively
`
`
`
`conformations are determined by the interactions of a few
`whose domains, the loops are connected to a /3-sheet framework
`
`
`
`
`
`
`residues at specific sites in the hypervariable regions and, for
`and affinity of the bind­
`
`structure is conserved
`
`
`
`
`certain loops, in the framework regions. Hypervariable regions
`
`
`ing sites are governed by the structures of the six hypervariable
`
`
`that have the same conformations in different immunoglobulins
`
`have the same or very similar residues
`
`
`Two models can be proposed for the relationship between
`of the amino-acid sequence of the anti­
`
`
`
`
`
`the amino-acid sequence and structure of the binding-site loops.
`
`
`body 01.3 showed that its hypervariable regions are the same
`
`
`
`In one model, different sequences produce different conforma­
`
`
`size as those in known structures and contain the same or similar
`tions for both the main chain and side chains of the loops.
`
`
`
`residues at the sites responsible for known conformations9• On
`
`
`
`Because hypervariable regions have different sequences in
`
`
`the basis of these observations the atomic structure of the VL-YH
`
`
`
`different antibodies, this model implies that each region adopts
`
`dimer of DI .3 was predicted before its experimental determina­
`
`
`
`
`a different conformation in different antibodies. In the other
`
`
`tion. Comparison of this predicted structure with the preliminary
`
`
`
`crystal structure showed that the conformations of four of the
`
`
`
`hypervariable regions had been predicted correctly; the confor­
`
`
`
`mation of L3 was significantly different from that predicted, and
`
`** Present address: The Squibb Institute for Medical Research, PO Box 4000,
`NATURE · VOL 342 · 21/28 DECEMBER 1989
`
`Princeton. New Jersey 08543-4000, USA.
`
`© 1989 Nature Publishing Group
`
`817
`
`1 of 7
`
`BI Exhibit 1049
`
`

`

`ARTICLES
`
`TABLE 1 Sequences and conformations of V K and VH hypervariable regions of known structure
`
`L1 Regionst
`
`Canonical
`Structure
`
`3
`
`4
`
`Protein
`
`26
`
`27
`
`28
`
`29
`
`30
`
`31
`
`32
`
`25
`
`33
`
`71
`
`J539
`HyHEL-5
`NQ10
`
`REI
`01.3
`HyHEL-10
`NC41
`
`McPC603
`
`4-4-20
`
`s
`s
`s
`
`s
`s
`s
`s
`
`s
`
`s
`
`s
`s
`s
`
`Q
`G
`Q
`Q
`
`Q
`
`s
`s
`s
`
`0
`N
`s
`0
`
`s
`
`s
`
`v
`v
`v
`
`I
`v
`
`s
`N
`R
`
`I
`H
`G
`s
`
`v
`
`K
`N
`N
`
`N
`
`H
`
`s
`y
`y
`
`v
`y
`N
`A
`
`y
`
`L
`M
`M
`
`y
`y
`y
`
`y
`y
`F
`y
`
`A
`A
`A
`
`A
`A
`A
`A
`
`s
`
`s
`
`s
`
`s
`
`G
`
`N
`
`N
`
`N
`
`K
`
`N
`
`G
`
`v
`
`Total no. of sequences known for L1 regions: human. 95; mouse, 299.
`1
`2
`4
`Canonical structure
`3
`Human sequences that fit (%)
`60
`5
`5
`Mouse sequences that fit (%)
`25
`10
`20
`
`15
`
`L2 Regions
`
`Canonical
`Structure
`
`Protein
`
`50
`
`51
`
`52
`
`48
`
`64
`
`REI
`McPC603
`J539
`01.3
`HyHEL-5
`HyHEL-10
`NC41
`NQ10
`4-4-20
`
`E
`G
`
`y
`0
`y
`w
`0
`K
`
`A
`A
`
`A
`A
`T
`v
`
`s
`s
`s
`T
`s
`s
`s
`s
`s
`
`G
`G
`G
`G
`G
`G
`G
`G
`G
`
`Total no. of sequences known for L2 regions: human, 69; mouse, 183.
`1
`Canonical structure
`Human sequences that fit (%)
`95
`Mouse sequences that fit (%)
`95
`
`L3 Regions
`
`Canonical
`Structure
`
`3
`
`Protein
`
`91
`
`92
`
`93
`
`94
`
`95
`
`96
`
`90
`
`REI
`McPC603
`01.3
`HyHEL-10
`NC41
`4-4-20
`NQ10
`
`J539
`
`HyHEL-5
`
`y
`0
`
`s
`
`s
`H
`
`w
`
`w
`
`w
`
`Q
`H
`w
`N
`y
`T
`s
`
`T
`
`G
`
`s
`s
`s
`s
`s
`H
`s
`
`y
`
`R
`
`L
`y
`T
`w
`
`v
`N
`
`N
`
`y
`L
`R
`y
`w
`w
`L
`
`Q
`N
`H
`Q
`Q
`Q
`
`Q Q Q
`
`Total no. of sequences known for L3 regions: human, 52; mouse, 152.
`2
`1
`3
`Canonical structure
`Human sequences that fit (%)
`2
`90
`Mouse sequences that fit (%)
`80
`1
`
`10
`
`Hl had a very different fold from that predicted9• (We report
`
`
`ruination of the sets of residues responsible for the observed
`
`
`
`
`
`
`below that the refined conformation of Dl.3 corresponds more
`
`
`
`conformations and (2) changes in the identity of residues at
`
`
`closely to the predicted structure.)
`
`
`
`other sites not significantly affecting the conformations of the
`
`
`An examination of the library of the known immunoglobulin
`
`
`
`canonical structures. The model can be tested, refined and
`
`
`sequences shows that many immunoglobulins have hyper­
`
`
`
`extended by using it to predict the atomic structures of binding
`
`
`variable regions that are the same size as those in the known
`
`
`sites in immunoglobulins before their structures have been deter­
`
`
`
`
`
`structures and contain the same or closely related residues at
`mined by X-ray crystallography.
`
`
`the sites responsible for the known conformations8. These
`
`
`We have now tested the canonical structure model by using
`
`
`observations indicate that for at least five of the hypervariable
`
`
`
`it to predict the structures of four immunoglobulins before their
`
`
`
`regions there is only a small repertoire of canonical main-chain
`
`structures had been experimentally determined. These
`
`
`
`
`conformations and that the conformation actually present can
`
`immunoglobulins are HyHEL-5 (ref. 16), HyHEL-10 (ref. 17),
`
`
`
`often be predicted from the sequence by the presence of specific
`
`
`NC41 (ref. 18) and NQlO (S.S., P.M.A. and R.J.P., manuscript
`residues.
`
`
`
`
`in preparation). The analysis of the amino-acid sequences of
`
`
`The accuracy of the canonical structure model for
`
`
`these immunoglobulins indicated that 19 of their 24 hypervari­
`
`
`
`immunoglobulin binding sites depends on (1) the correct deter-
`
`
`able regions should have conformations close to known canoni-
`
`NATURE · VOL 342 · 21/28 DECEMBER 1989
`
`878
`
`© 1989 Nature Publishing Group
`
`2 of 7
`
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`
`

`

`ARTICLES
`
`94
`
`R
`R
`R
`R
`R
`R
`R
`G
`
`R
`N
`
`30
`
`s
`s
`s
`s
`s
`s
`s
`
`T
`
`T
`
`31
`
`s
`0
`s
`0
`
`0
`
`K
`G
`
`N
`
`N
`0
`
`32
`
`y
`y
`y
`y
`y
`y
`0
`
`F
`
`34
`
`M
`v
`y
`
`M
`
`M
`
`I
`M
`M
`M
`
`0
`
`w
`
`54
`
`55
`
`71
`
`H
`0
`
`T
`
`G
`
`G
`G
`
`G
`G
`
`G
`
`s
`G
`s
`(•)
`s
`(•)
`s
`s
`s
`y
`K
`(•)
`N
`y
`
`L
`
`A
`
`R
`R
`R
`
`R
`R
`
`Hi Regions+
`
`Canonical
`Structure
`
`1'
`
`Protein
`
`KOL
`
`McPC603
`
`J539
`01.3
`HyHEL-5
`NC41
`NQ10
`4-4-20
`
`NEW
`HyHEL-10
`
`26
`
`27
`
`28
`
`29
`
`G
`G
`G
`G
`G
`G
`G
`G
`
`G
`G
`
`y
`y
`s
`
`F
`F
`
`0
`
`I
`
`0
`T
`T
`
`s
`T
`T
`T
`
`s
`
`Total no. of sequences known for H1 regions: human. 50; mouse, 321.
`1
`Canonical structure
`Human sequences that fit (%)
`50
`80
`Mouse sequences that fit (%)
`
`H2 Regions§
`
`Canonical
`Structure
`
`Protein
`
`52a
`
`NEW
`01.3
`HyHEL-10
`
`HyHEL-5
`NC41
`
`KOL
`J539
`NQ10
`
`T
`
`p
`s
`
`0
`
`McPC603
`4-4-20
`
`N
`N
`
`K
`K
`
`p
`
`G
`
`2
`
`3
`
`4
`
`Total no. of sequences known for H2 regions: human, 54; mouse. 248.
`Canonical structure
`1
`3
`4
`2
`Human sequences that fit (%)
`15
`1
`40
`15
`Mouse sequences that fit (%)
`15
`40
`5
`20
`
`53
`
`y
`y
`
`G
`
`G
`N
`
`0
`0
`G
`
`y
`
`N
`
`The residues listed here (single-letter code) are those that form the hypervariable regions and those in the framework regions that are important for the observed conformations
`8
`. The hypervariable regions are taken as those outside the framework {:l-sheet8
`of these regions
`. Except for H2. they are similar to, but not identical with the regions that show
`
`or canonical structure, are adjacent. The canonical structure numbers used below refer to the conformations shown in Fig. 1. The residues in the hypervariable and framework
`regions that are mainly responsible for these conformations8
`are indicated by an asterisk. The classification and sequence requirements of the H2 conformations have been
`
`also give the percentage of these sequences that are the same size as the known canonical structures and have the same residues at the positions marked by an asterisk.
`
`8
`. The remaining residues form a turn or loop on the surface (Figs 1 and 4). The ends of the long loops
`26-19 and 32 packed against the framework in the same conformation
`have some flexibility. There are another 25% of the human sequences and 20% of the mouse sequences that have one more residue than structure 2. or one fewer than structure
`4, and whose sequences satisfy the requirements listed above. It is expected that these differ only in the conformations of the tips of the surface loops.
`
`high sequence variations and which Kabat et a/.26 use to define hypervariable regions. The sequences are grouped so that those that have the same main-chain conformation.
`revised in the light of work described here and elsewhere28. For each hypervariable region the number of human and mouse sequences listed by Kabat et a/.26 are given. We
`t Canonical structure ·4 is illustrated in Fig. 4. Although the size of the known L1 structures varies between 6 and 13 residues. they have closely related folds with residues
`+The H1 hypervariable regions with canonical structure 1 have very similar conformations: the r.m.s. differences in the coordinates of their main-chain atoms are 0.3-0.8 A.
`and 56-63 do not differ significantly8 (Fig. 1b). (•). The residues at positions 55 or 54 in the canonical structures 2, 3 and 4 have residues with positive values for q, and i/J,
`
`The Hi regions in NEW and HyHEL-10 only partly satisfy the sequence requirements for structure 1 and have a distorted version of its conformation.
`§The H2 region here comprises residues 52a-55. The region with high sequence variation is 50-65 (ref. 26). In the known structures the main-chain conformation of 50-52
`
`and usually, but not in all cases, Gly, Asn or Asp is found at these sites. For a sequence to match that of canonical structure 2, 3 or 4 the presence of these residues at sites
`54 or 55 is required.
`
`in Table l. Each hypervariable
`
`cal structures. We then compared the predicted structures of
`
`
`
`region in the immunoglobulins
`
`
`
`these hypervariable regions with the subsequently determined
`
`
`of unknown structure was examined to determine ( 1) whether
`
`
`
`structures. Another immunoglobulin structure, 4-4-20 (ref. 19)
`
`
`it has the same size as any homologous hypervariable region of
`
`
`
`has recently been reported. We did not have the opportunity to
`
`
`
`known structure and (2) whether its sequence contains the set
`
`
`
`predict the structure of 4-4-20 before its experimental determina­
`
`
`
`of residues responsible for a known conformation. Except for
`
`
`tion, and we discuss here only how its hypervariable regions
`
`
`
`L3 in HyHEL-5, all the light-chain regions correspond to a
`
`
`have the conformations expected from the known canonical
`
`
`
`known canonical structure, as do all the HI regions and the H2
`
`
`structures. Also, we report that the refined conformation of Dl.3
`
`region in HyHEL-10 (Table I). The conformation predicted for
`
`
`
`(ref. 20) corresponds more closely to the predicted structure.
`
`the H2 regions in NC41 and HyHEL-5 was based on the analysis
`
`
`
`of the H2 region in the preliminary structure of J539 (ref. 8).
`
`In all three of these antibodies the H2 region is a four-residue
`Model building procedure
`
`
`turn with Gly at the fourth position and the predicted conforma­
`The main-chain conformations of the hypervariable regions in
`
`
`
`
`
`tion is that almost always found for such turns21• (Below we
`are shown in Fig.
`
`
`
`
`present a more accurate analysis of H2 regions.) For H3 regions
`
`1. The residues responsible for these conformations are listed
`
`
`in HyHEL-5, HyHEL-10, NC41 and NQIO, no prediction of
`
`the V K and VH domains of known structure
`NATURE · VOL 342 · 21/28 DECEMBER 1989
`
`
`
`
`
`© 1989 Nature Publishing Group
`
`879
`
`3 of 7
`
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`
`

`

`ARTICLES
`
`of the hypervariable and framework regions, one known V L and
`
`
`conformation could be made on the basis of the known canonical
`
`
`
`one known VH structure were taken as the starting points­
`structures.
`
`
`
`parents-for the model of the predicted structure. If the confor­
`
`The sequences of the V L and VH domains were compared to
`
`
`
`mation predicted for a hypervariable region was not present in
`
`see which of the known framework structures have sequences
`
`
`
`the parent, but was present in another known structure, the
`
`close to those of the unknown structures. From the comparisons
`
`
`hypervariable region in the parent was replaced by that in the
`
`
`
`other structure. Side chains in the parent that were different
`
`
`from those in the unknown structure were replaced22•23 and the
`
`
`
`resulting model subjected to a very limited energy refinement24•
`
`a
`
`2.5 A, 20%; HyHEL-
`
`3.0 A, 24%; and NC41-neuraminidase, 2.9 A,
`����
`O" �1--C L3 �H2
`N
`VL
`
`HyHEL-5, HyHEL-10 and NC41 hypervariable regions
`
`The atomic structures of Fab fragments of immunoglobulins
`
`HyHEL-5, HyHEL-10 and NC41 in complexes with their protein
`
`antigens were determined by X-ray crystallography16-18• The
`
`
`
`
`resolution of the X-ray data used to determine the structures
`
`
`and value of the residual (R) after refinement are (complex,
`
`
`resolution, R value): HyHEL-5-lysozyme,
`10-lysozyme,
`19%. These structures have been determined at medium resol­
`
`
`
`
`ution. The tracing of the polypeptide chain of the hypervariable
`
`
`
`
`regions is unambiguous, although the orientation of some of
`
`VH
`
`b
`
`L1
`
`Antibo d y
`
`Antigen binding site
`
`VK
`
`lie
`2
`
`26
`
`3
`
`3
`
`L3 �;�.
`90Lv
`GI�� .. .-!
`··'�-97
`··
`1
`
`·····
`
`2
`
`5h 52
`Ile41J(0 •
`
`L2
`
`VH
`
`H2
`
`880
`
`2
`
`3
`
`4
`
`© 1989 Nature Publishing Group
`
`NATURE · VOL 342 · 21128 DECEMBER 1989
`
`FIG. 1 a, Antigen-binding
`
`sites of immuno­
`globulins are formed by six loops of poly­
`
`peptide, three from the VL domain L1, L2
`and L3 and three from the VH domain H1, H2
`
`and H3 (wavy Jines). These loops are attached
`
`to strands (D) of a conserved {3-sheet. b,
`regions of V K and VH domains. In each drawing
`
`
`
`Canonical structures for the hypervariable
`
`the region is viewed so that the accessible
`surface is at top and the framework region
`
`
`below. The main-chain conformation and some
`of the side chains that determine this confor­
`
`
`mation are shown. For the definition of the
`
`hypervariable regions used here, see Table 1.
`
`The immunoglobulins in which the different
`
`
`canonical structures occur are listed in Table 1.
`
`Phe29 �
`
`H1 �\�'
`� 52� .·}
`.. 56
`�--.....
`50�··.· ..
`58
`
`4 of 7
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`ARTICLES
`
`to each other by 1-2 A, and the ends of
`
`
`
`the peptide groups is uncertain. Most side chains are unequivo­
`ing. First, the predicted structures were built using parent V
`
`
`
`
`
`domains that have some residues in the framework and V L-VH
`cally placed.
`
`
`interface that are different from those in the final predicted
`
`
`
`Figure 2 shows the predicted and observed structures of each
`
`
`
`structure. These differences have small but significant effects on
`
`
`
`
`of the hypervariable regions, superposed by a least-squares fit
`
`
`
`the main-chain structure of the individual domains and the way
`
`of their main-chain atoms. Table 2a gives, for each predicted
`
`
`
`
`
`and observed hypervariable region, the r.m.s. difference in posi­
`
`
`they pack together2•25'26• Second, differences between the pre­
`
`tion of the main-chain atoms.
`
`
`dicted and observed structures could arise from the effects of
`
`
`
`the association with the antigen 18• In other proteins, ligand
`
`
`
`The main-chain conformations of the predicted and observed
`
`binding can result in the movement of close-packed segments
`
`
`
`hypervariable regions are very similar (Fig. 2, Table 2a ). The
`
`of polypeptide relative
`
`
`only exception is the Hl region of HyHEL-5. Although the
`loops are able to move somewhat more27.
`
`
`
`
`observed and predicted conformations of residues 26-29 and
`
`
`
`The differences in the positions of the predicted and observed
`
`32 are the same, residues 30 and 31 are in quite different
`
`H2 regions in HyHEL-5 and NC41 (Table 2b) are larger than
`
`
`
`positions. The recently refined structure of Fab 1539 (T. N. Bhat,
`
`
`
`expected from these factors. The interactions that the H2 regions
`
`
`E.A.P. and D.D., manuscript in preparation) shows that these
`
`make with the rest of the VH domain were therefore examined.
`
`
`differences were inherited as a result of an error in the original
`
`
`determination of the 1539 structure used as the parent for this
`Residue 71 and position and conformation of H2
`
`
`
`
`region. Rebuilding the predicted model with the refined 1539
`
`At the same time as the structures of HyHEL-5 and NC41
`
`
`
`
`structure puts residues 30 and 31 in the correct position and
`
`
`
`
`became available, the refinement of the atomic structure of 1539
`
`
`gives an r.m.s. difference between the predicted and observed
`
`
`was completed (T. N. Bhat, E.A.P. and D.D., manuscript in
`Hl regions
`
`
`
`preparation). The conformation of H2 in the refined structure
`
`
`Given the medium resolution of the structures used to derive
`is not like that in HyHEL-5 and NC41 but is the same as that
`
`
`the models and of the experimental structures, the agreement
`
`
`in KOL. This was quite unexpected. The main determinant of
`
`
`
`of the predicted and observed loop conformations is excellent.
`
`
`
`the conformation of small turns is usually the position of glycine
`Relative positions of the hypervariable regions
`
`
`residues21: in KOL, Gly occurs at position 54, and in 1539,
`
`HyHEL-5 and NC41, Gly occurs at position 55.
`
`
`
`Figure 3 shows the positions of the hypervariable regions relative
`
`
`An examination of the environments of the H2 regions28
`
`
`to each other and to the framework for the predicted and
`
`shows that in KOL and 1539 the side chain of framework residue
`
`
`
`observed structures. To produce this figure the predicted and
`
`
`Arg 71 packs between, and forms hydrogen bonds to, HI and
`
`
`
`
`observed structures were superposed by a least-squares fit of
`
`H2. In HyHEL-5, residue 71 is Ala, and here the cavity that
`
`
`
`
`framework residues. In Table 2b, the differences in position of
`
`would be created by this smaller side chain is filled by the
`
`
`the hypervariable regions are reported.
`
`
`
`insertion of a residue from the H2 region-Pro 52a. In KOL
`
`
`
`Small differences in the relative positions of the hypervariable
`
`
`and 1539 the side chain at position 52a is on the surface. The
`
`
`
`
`regions in the predicted and observed structures might be expec­
`
`
`
`relative movement of position 52a involves a change in the
`
`
`ted because of two factors not corrected for in the model build-
`
`of 0.6 A.
`
`ffy M�r �,
`
`HyHEL·5L1
`
`HyHEL-10l1
`
`NC41L1
`
`NO•OL1
`
`Hy HEL-5 L2
`
`Hy HEL-10 l2
`
`NC41 L2
`
`N010 L2
`
`HyHEL-10l3
`
`NC41 L3
`
`N01 0 L3
`
`Hy HEL-5 H1
`
`Hy HEL-10 H1
`
`NC41 H1
`
`N010 H1
`
`FIG. 2 The predicted
`
`Table 2a. Predicted
`
`(broken line) and observed
`
`
`(continuous line) conformations of the hypervari­
`
`
`able regions. The structures have been super­
`
`posed by a least-squares fit of their main-chain
`atoms. Residue numbers and the r.m.s. difference
`
`in position of the superposed atoms are given in
`
`and observed side-chain con­
`formations are shown for al I regions except H1
`
`and L3 in NQ10 where they obscure the main chain.
`
`
`After our prediction of the NC41 structure several
`
`revisions were made to the sequence and some
`
`of the differences can be seen here.
`
`Hy HEL-5 H2
`
`Hy HEL-10 H2
`
`NC41 H2
`
`N010 H2
`
`NATURE · VOL 342 21/28 DECEMBER 1989
`
`© 1989 Nature Publishing Group
`
`881
`
`5 of 7
`
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`

`ARTICLES
`
`TABLE 2 Differences in structure of predicted and observed hypervariable regions
`
`(a) Differences in local conformation (A)
`
`Hypervariable
`region
`
`R.m.s. difference in atomic positions of main-chain
`atoms after optimal superposition
`NC41
`HyHEL-10
`
`NQ10
`
`HyHEL-5
`
`L1:26-32
`L2:49-53
`L3:90-97
`H1:26-32
`H2:52-56
`
`0.8
`0.9
`
`1.4
`1.1
`
`1.1
`0.8
`0.3
`1.3
`1.0
`
`0.7
`0.4
`0.5
`0.9
`0.7
`
`0.4
`0.9
`0.6
`0.3
`0.4
`
`(b) Differences in position relative to framework (A)
`superposition of frameworks residues (A)
`
`Range of differences in positions of Ca atoms after
`
`Hypervariable
`region
`
`Original
`prediction
`
`New structure
`library
`
`Ll
`L2
`Hl
`H2
`
`Ll
`L2
`L3
`
`H1 H2
`L3 Hl
`
`Ll
`L2
`
`H2
`
`2.0-3.8
`1.6-1.6
`0.8-4.1
`3.0-7.2
`
`0.7-1.6
`0.6-.13
`0.8-1.5
`1.3-3.5
`0.6-2.9
`
`1.4-2.4
`1.1-1.8
`1.6-2.3
`1.3-2.0
`2.1-4.4
`
`0.8-2.1
`1.2-2.3
`0.7-2.1
`0.5-2.1
`
`1.5-2.6
`1.0-2.0
`1.8-3.0
`1.0-2.1
`0.4-1.9
`
`Protein
`
`HyHEL-5
`
`HyHEL-10
`
`NC41
`
`NQ10
`
`Ll
`L2
`L3
`Hl
`H2
`
`0.4-2.7
`0.5-1.4
`0.6-1.5
`0.6-1.2
`0.6-0.9
`
`analysis described above, canonical structure 2 was expected
`
`
`
`
`
`because of the Arg at position 71 (Table 1).
`Recently a crystal structure has been determined for NQlO
`
`
`
`
`
`
`
`
`(S.S., P.M.A. and R.J.P., manuscript in preparation). This struc­
`
`ture is determined to a resolution
`
`
`residual is 21 %. The tracing of the chain in the hypervariable
`
`
`regions is unambiguous. Figure 2 shows the predicted and
`
`
`
`observed structures of each of the hypervariable regions, super­
`
`
`posed by a least-squares fit of their main-chain atoms. Table 2a
`
`
`
`gives the r.m.s. differences in position of the main-chain atoms.
`
`
`
`Figure 3 shows the relative positions of the predicted and
`
`
`observed hypervariable regions.
`There is close agreement between the predicted and observed
`
`
`
`
`
`
`main-chain conformations of the hypervariable regions: the
`
`
`r.m.s. differences in position
`2a ). There is also close agreement in the relative positions of
`
`
`
`
`the hypervariable regions ( Fig. 3 and Table 2b ). No residues
`and all but two are within
`
`of 2.8 A and the present
`are between 0.3 and 0.9 A (Table
`differ by more than 2.7 A in position
`1.1 A. Recently,
`
`the atomic structure of antibody Dl.3 complexed
`
`
`
`
`
`with lysozyme has been refined20. The comparison of the pre­
`
`
`
`
`liminary experimental structure with the prediction had shown
`
`
`
`
`differences for two hypervariable regions9. The L3 regions had
`
`
`
`differences associated with Pro 95 having a cis-peptide in the
`
`
`
`
`predicted structure and a trans-peptide in the observed. The Hl
`
`
`
`regions differed because the predicted structure had this region
`
`
`folded into framework, whereas in the observed it folded out
`
`
`
`into the solvent. The refinement of the experimental structure
`
`
`
`has resulted in the rebuilding of these two regions20, and their
`
`
`conformations now agree with the original predictions.
`
`of 2.7 A (ref. 19). The amino­
`
`hypervariable regions in the original predictions and the observed structures of b are
`Superposition of a are illustrated in Fig. 2. The differences in the positions of the
`HyHEL-5 model using the refined VL J539 and the VH NC41 structures, and rebuilding
`the NC41 model using VL McPC603 and VH HyHEL-5:
`
`Canonical structures in immunoglobulin 4-4-20
`
`
`The atomic structure of immunoglobulin 4-4-20 has recently
`
`been determined at a resolution
`
`acid sequence of the hypervariable regions and associated
`framework sites of 4-4-20 are given in Table 1. Four of the
`
`
`hypervariable regions, L2, L3, Hl and H2, have the size and
`
`
`
`the residues at specific sites that clearly indicate particular
`
`
`canonical structures (see Table 1). These four hypervariable
`
`
`
`
`regions do have the expected main-chain conformations. fn Fig.
`conformation of H2. It also tilts the H2 loop so that the positions
`
`
`
`
`4 the four hypervariable regions, superposed on examples
`
`of residues at the top of the loop in HyHEL-5 differ in position,
`
`
`of the same canonical structures taken from other immuno­
`relative
`
`
`globulins, are shown. The r.m.s. difference in the atomic
`
`
`
`residue at position 71 is Leu and that at position 52a is Thr,
`and the shift is not as large as that in HyHEL-5.
`This analysis implies that the conformation and position of
`
`
`
`
`
`
`
`four residue H2 regions are determined by the packing against
`
`
`the VH framework and by the identity of the residue at position
`
`
`
`71 in particular, and not by the position in the sequence of H2
`
`
`of Gly (or Asn or Asp) as was believed previously8. Thus VH
`
`domains of HyHEL-5 and NC41 should provide better parents
`
`for each other than the other structures do, for they contain
`
`
`
`similar determinants for the position of H2. The predicted
`
`
`
`structure of HyHEL-5 using the observed structure of VH of
`
`
`NC41 and the predicted structure of NC41 using the observed
`
`
`structure of VH of HyHEL-5 were thus rebuilt. In these new
`
`
`
`predicted structures the large differences between the predicted
`
`
`
`and observed positions of H2 are not present: most residues
`
`Hypervariable regions of NQ10 and 01.3
`regions in the predicted of the hypervariable FIG. 3 The relative positions
`
`
`
`The amino-acid sequence of the hypervariable regions and
`
`
`
`
`
`and observed structures. The positions of the hypervariable regions shown
`
`associated framework sites of NQlO are given in Table 1. For
`
`are those given by the superposition of the VL-VH framework regions of
`
`
`
`five of the hypervariable regions, the size and the residue con­
`
`
`the observed and predicted structures. The Ca atoms of residues in the
`
`
`
`
`
`servation at the relevant sites clearly indicate particular canoni­
`
`observed structure are joined by solid lines and those in the predicted
`
`cal structures, and a model of the VL-VH dimer of NQlO was
`
`
`
`structure by broken lines. The size of the differences in position are given
`
`made using the procedure described above. For H2, after the
`
`882
`
`in Table 2b.
`
`© 1989 Nature Publishing Group
`
`NATURE · VOL 342 · 21/28 DECEMBER 1989
`
`shown in Fig. 3. The predictions with a new structure library involve rebuilding the
`
`see text
`
`to those in KOL and 1539, by �4.5 A. In NC41, the
`
`L2(_;
`
`Hy HEL · 5
`
`differ by no more than 2.1 A and none differs by more than
`3.0 A (Table 2b ).
`
`
`
`
`
`The results of this analysis were used in the prediction of the
`
`
`structure of the H2 region of the antibody NQlO.
`
`Hy HEL -10
`l2v
`f Q\
`L3
`L1
`
`NC41
`
`7. NQ10
`L2 �
`
`j"'
`0H2
`jH1
`j H2
`
`6 of 7
`
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`
`

`

`ARTICLES
`
`the canonical structure model. It implies that a good estimate
`
`
`
`
`
`can be made of the extent to which hypervariable regions in
`
`
`
`other immunoglobulins have main-chain conformations close
`to those that are now known. This can be done by inspecting
`
`
`the known sequences to see whether their hypervariable regions
`
`
`
`correspond in size to the known canonical structures, and
`
`whether they contain one of the sets of residues that produce
`
`the observed conformations.
`
`
`In Table 1 we give the results of examining the immuno­
`
`
`globulin sequences collected
`and -70% of the H 1
`
`the hypervariable regions
`
`
`
`and H2 regions in VH domains, are expected to have conforma­
`
`
`tions close to those found in the immunoglobulin structures now
`
`
`known. These estimates are conservative in that they include
`
`
`only hypervariable regions that match the sequence require­
`
`
`
`ments exactly, that is, those that contain the particular residues,
`
`
`
`or any combination of the small range of closely related residues,
`
`that are found in the known structures at the sites marked by
`asterisks
`
`by Kabat et al. 29. About 90% of
`in V K domains,
`
`in Table I.
`
`H1
`·,
`··· ...
`.
`56
`·. �:. ;:
`
`··--::
`52 .
`
`.
`
`.
`
`·:-,
`
`.
`
`.. ·.·.� .............
`..... .
`.h ......
`H2 Fab
`
`4 - 4 -20
`
`L1
`
`L2
`
`L3
`
`of 2 A or less.
`
`0
`
`Discussion
`The wide occurrence of the known canonical structures, the use
`
`
`
`
`
`of the much larger library of parent structures that will soon
`
`
`
`become available, and the more detailed understanding of the
`
`
`
`determinants of structure that will emerge from the analysis of
`
`
`the differences between predicted and observed structures,
`
`
`should allow, for many immunoglobulins, the prediction of the
`
`
`
`
`structure of five of the hypervariable regions with accurate local
`
`conformation and errors in position
`4-4-20. The observed FIG. 4 The hypervariable regions in immunoglobulin
`
`
`
`
`Predictions of the structures of the H3 regions in the
`
`
`conformations of the hypervariable regions in 4-4-20 are drawn with con­
`
`tinuous lines. They are superposed on hypervariable regions from other
`
`
`immunoglobulins discussed here could not be made on the basis
`
`
`immunoglobulins that have the same canonical structure-L2 and L3 from
`
`
`
`of the previously known structures. It was possible to predict
`
`
`
`correctly the H3 conformation in immunoglobulin 0 1.3 (ref. 9).
`
`
`
`
`
`In general, however, the various genetic mechan