THK JOURNAL. OF BiOLOGICAL CHEMISTRY
`Vol. 253, No. 2, Issue of January 25, pp. 585-597, 1978
`Printed in U.S.A.
`
`From the Department of Biophysics, The Johns Hopkins University School of Medicine, Baltimore,
`
`(Received for publication, July 7, 1977)
`
`
`
`The three-dimensional structure of the Fab fragment
`are discussed
`
`
`in relation to the genetic control and physiological function of
`
`"model building" and "real space" procedures. By these
`
`immunoglobulins.
`It is generally accepted (see review in Ref. 1) that electron
`
`
`techniques, the correlation between the amino acid se­
`multiple isomorphous
`
`
`density maps calculated by multiple isomorphous replacement
`
`replacement Fourier map has been optimized. The average
`
`shift of all atoms during real space refinement
`
`Preliminary Refinement and Structural Analysis of the Fab
`Fragment from Human Immunoglobulin New at 2.0 A
`Resolution*
`FREDERIC!< A. SAUL, L. MARIO AMZEL, AND ROBERTO J. POLJAKt
`Maryland 21205
`Fab New.1 Some features of the refined structure
`from human myeloma lgG New has been refined using
`quences and the 2.0 A resolution
`was 0.62 A. A
`techniques contain significant errors which may lead to impre­
`list of the refined atomic coordinates for the 440 amino acid
`amino acid side chain atoms, bond angles, r/l and ijl values, cis
`cise determination of structural details such as the location of
`prepared using the refined coordinates show a distribution
`project was undertaken w1th the aim of obtaining more
`or trans character
`etc. This refinement
`which can be appHed to structural studies
`of other immunoglobulins and Fab · hapten complexes (2). The
`to the predomi­
`of q,, 1/.1 angular values which corresp()nds
`accurate coordinates
`starting atomic coordinates were those of the structure previ­
`
`nant JI-pleated sheet conformation present in the structure.
`(3, 4) obtained using multiple isomorphous
`
`The structures of the homology subunits V11, VL, Cnl, and
`
`CL were superimPosed by pairs and quantitatively com­
`ously described
`of two
`were @bserved between V H
`pared. The closest similarities
`
`
`heavy atom replacements. Since the complete refinement of
`the structure of Fab New is a complex undertak ing, we
`
`and VL and between Ciil and CL. Amino acid sequence
`refinement techniques. In the first step we have
`
`
`alignments obtained from this structural superposition are
`present here initial results obtained after application
`served between CHI(')' heavy chain) and C1, (A. light chain).
`given. The closest sequence homology in Fab New is ob­
`consecutive
`were adjus.ted to impose standard
`
`
`applied a "model building'' procedure (5) in which the mea­
`procedure (6, 7) was used to optimize the correlation between
`In addition, there is considerable homology between the
`
`sured atomic coordinates
`bond lengths and bond angles. In a second step a "real space"
`variable and constant regions.
`The distances of close contacts between the homology
`a distances; 1.2 times their
`
`subunits of Fab New have been determined. The closer
`the Fab New model and the multiple isomorphous replace­
`nature of the immunoglobulin
`an improved model which has been used to compare the
`contacts,
`those between atoms at
`ment, electron density Fourier map.
`van der Waals radii, are analyzed in relation to the constant,
`
`The coordinates obtained by these P'rocQdures have provided
`
`variable, and hypervariable
`
`sequence positions at which they occur. Most of the residues
`tertiary structure of the homology subunits, to calculate
`Fab, and to re-examine
`
`interatomic contacts that define the quaternary structure of
`which determine the closer contacts between V11 and VL and
`
`the conformation of the combining
`
`between CH 1 and Ci. are structurally homologous and highly
`
`conserved or conservatively replaced in immunoglobulin
`site.
`sequences.
`measured on the 2 A (nominal) resolution model previously de­
`METHOOS
`Measurement of Model Coordinates -Atomic coordinates were
`The relation between idiotypic determinants, antigen
`
`
`
`combining site and hypervariable regions, is discussed in
`scribed (4). A two-pointer device was used for this purpose: a
`terms of the refined model.
`horizontal pointer (50 inches long) was brought to touch atom
`centers by displacing the device on the base of the model, adjusting
`crystallographic refinement and a list of atomic coordinates of
`fixed poin�r of equal length gave the x, y while a second, parallel,
`In this paper we present the results of a preliminary
`the height of the pointer aloni;r a i;rraduated scale (z coordinate)
`
`of the model. The use of a level and
`* This work was supported by Research Grant AI 08202 from the
`leveling screws at the base of the two-pointer device was essential
`National Institutes of Health and by Researeh Grants NP-141B and
`for obtaining reproducible coordinates. While these measurements
`were made, the image of the model and the atom centers were
`This article must therefore be hereby marked "advertisement" in
`accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
`' The abbreviations used for immunoglobulins, their polypeptide
`t Recipient of United States Public Health Service Research
`chains, and fragments are as recommended in (1964) Bull. W H 0
`30, 447.
`
`residues in the structure is given. Ramachandran plots
`
`of proline residues,
`
`IM-105C from the American Cancer Society. The costs of publication
`of this article were defrayed in part by the payment of page charges.
`
`coordinates on a grid at the base
`
`Career Development Award AI-70091.
`
`585
`
`1 of 14
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`BI Exhibit 1083
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`

`

`586
`
`Structural Refinement of Fab New
`
`TABLE II
`
`projected on the corresponding sections of the Fourier map using an
`optical comparator (8) to verify that their location and the coordinate
`values corresponded with the Fourier map.
`Model Building Procedure -The set of measured coordinates for
`the 3185 non-hydrogen atoms in the structure provided the starting
`
`point for this procedure. In general, these coordinates are subject to
`
`errors due to measurement uncertainty and to mechanical deforma­
`tions of the skeletal brass model. Consequently, the mathematical
`model building procedure of Diamond (5) programmed for a digital
`computer was used in order to impose standard bond lengths and
`bond angles in the model. The measured coordinates were used to
`provide a guide point for each (non-hydrogen) atom in the structure.
`Some conditions used in this part of the refinement process are given
`
`in Table I. All the varied angles are dihedral; the r hs-<:<>-<:l angle
`
`Conditions of real space refi.nernent•
`
`Zone length:
`6
`Margin width:
`Fixed atomic radii: 1.4 A
`
`5
`Relative atomic weights; C:6, 0:7, N:8, S:l6
`x: 3.2
`"'· ijJ: 4.0
`
`Relative softness of angular parameters that were allowed to vary:
`
`A,.,.,n
`
`Amir/A.max
`
`Filter levels:
`
`0.0001
`Sca�e factor and background
`0.01
`0.01
`0.001
`Translational refinement
`0.001
`0.001
`Rotational refinement•
`Electron density map grid; 111.43/160, 56.68/80, 90.30/130 along cell.
`edge's
`
`• SP.e Diamond (6, 7) for definition of terms.
`•Nonlinear constraints were used to preserve chain continuity.
`
`1). Many carbonyl groups of the main polypeptide chain can
`be reliably positioned (Fig. 2). In general, coordinates of atoms
`in regions of strong, well defined electron density converged
`rapidly in the first cycle of refinement and moved very little
`in subsequent cycles. Atoms in regions of lower density
`converged more slowly. A few residues
`in poorly defined
`regions of low density showed little convergence, although
`movement of main chain atoms tended to be smaller than
`their total shift from the starting coordinartes was small. The
`those of side chains, presumably due to their better defined.
`
`electron density and to grearter constraints on their positions.
`The progress of refinement was checked after each cycle by
`inspecting the fit of atoms whose shifts were substantially
`greater than the average. Some side chain groups which had
`been shifted by the Diamond model building procedure were
`
`moved back to their original positions by real space refine­
`than 1 A occurred for three consecutive amino acid residues
`(Gly 166, Val 167, and His 168) in the CHI region. Inspection
`
`ment. ln the fifth cycle ofrefinement, an average shift greater
`
`of the position of these atoms showed that they had moved to
`a conformation that appears to be in better agreement with
`the electron density map than the original model. The coordi­
`nates for all atoms of Fab New after refinement, given in the
`"Appendix," are filed with the Protein Data Bank at Brook­
`haven National Laboratory. No major features of the map
`remain unexplained, although a number of possible solvent
`molecules are found on the surface of the molecule. The
`conventional R factor,2 based on F, obtained with the coordi­
`nates. in "Appendix" and an overall temperature factor (B =
`ment approximations outlined in Table 11 and the fact that no
`18.0), is R = 0.46. This value is reasonable given the refine­
`
`solvent atoms were included in the model. Further refinement
`using observed structu.re factors and calculated phases is
`currently underway.
`The S-S distances in the five Fab disulfide bonds were
`allowed to vary without constraints. At the end of the refine­
`
`ment these distances were found to be: V H• 2.00 A; V L• 2.46 A;
`C.,1, 2.30 A; Ci.. 2.30 A; C11l-C1., 2.43 A.
`Ramachandran Plots
`'R = l I F0 -Fe I /lF0, where F0 is observed structure amplitude
`The Ramachandran plots of the V L and C1. homology sub-
`(-B sin' 0/A 2).
`3 The isotropic temperature factor (B) used fo the expression exp
`
`and F< is calculated structure amplitude.
`
`was allowed to vary since this condition gave a much closer correla­
`tion with input coordinates without introducing large distortions in
`the idealized, model-built geometry of the molecule. Residues for
`which the model-built coordinates differed considerably from the
`ured and checked for correspondence with the Fourier map. These
`input coordinates or which had an abnormal r0 value were remeas­
`of coordinates or to distortions of the brass model. When necessary
`discrepancies could always be traced to errors in the measurement
`ITo - 109.3°1> for the 440 residues in th1> model-built
`1:itr0(tirr0 =
`structure was 5.43°. Pro 151 in C., I was built and refined in the cis
`nates for all non-hydrogen atoms in the st.ruduri. wa!< 0. 2 A.
`conformation. The root mean square shift from the initial coordi­
`as a starting set for real space refinement (6). The 2.0 A electron
`at intervals of 1/160 along x, (a = ll 1.43 A), 1/80 along y (b = 56.68
`Al, and 1/130 along z (c = 90.30 A) in sections of constant y. A
`
`the coordinates were measured again nftcr rebuilding distorted
`regions and the remeasured coordinates were submitted to the model
`building procedure as guide points. The final average value of
`
`Real Space Refinement -The model-built coordinates were used
`
`density map used for the automatic fitting of the atomic coordinates
`was calculated using multiple isomorphous replacement phases as
`described before (3, 4). The electron density function was: calculated
`
`computer program incorporating a fast-Fourier transform algorithm
`was used for this calculation. Five cycles of real space refinement
`were carried out using the conditions defined in Table IL Values of
`atomic radii for carbon, nitrogen, oxygen, and sulfur that gave
`fastest convergence in trials using a small part of the structure were
`adopted and kept constant during refinement. Progress in the
`
`refinement process was followed by inspection of the root mean
`square shifts in coordinates, shifts in the coordinates of individual
`atoms, and adjustments of amino acid scale factors. Unusually large
`
`shifts in coordinates were checked using the optical comparator and
`the Fourier map. All refinement calculations were carried out using
`computer programs implemented at the Brookhaven National Lab­
`oratories.
`
`RESULTS AND DISCUSSION
`The root mean square shifts of atomic coordinates after five
`cycles of real space refinement converged to an average value
`
`of 0.09 A. The values after each cycle were: 0.46 A, 0.20 A, 0.13
`A, 0.10 A, and 0.09 A. The average shift of all atoms during
`real space refinement was 0.62 A. In most parts of the
`
`molecule, the coordinates after refinement are in very good
`agreement with the features of the electron density map (Fig.
`
`TABLE I
`Conditions of model building refinement•
`residues were used. In addition, r(Ca CfJ Cy) was allowed t-0 vary in
`Parameters varied were: q,, o/J, x:, T (N Ca C). Flexible proline
`Filter constants
`Diamond (7).
`Probe
`c.
`C,
`
`Cys, His, Phe, Trp, and Tyr residues. A list of the sources of the
`amino acid groups used in model building is given in Table I of
`
`Lenfuh
`(resi ues)
`
`I
`2
`3
`
`1
`2
`3
`
`0 See Diamond (5) for definition of terms.
`
`0.1
`0.1
`0.1
`
`10-•
`10-•
`10-•
`
`2 of 14
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`BI Exhibit 1083
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`

`

`
`
`Structural Refinement of Fab New
`
`587
`
`Stereo view of some main chain and side chain
`Stereo view of some amino acid residues of Fab
`New superimposed on the corresponding
`density of the multiple
`
`imposed on the corresponding electron density of the multiple
`
`isomorphous replacement map. Carbonyl groups of the main poly­
`map.
`peptide chain are clearly seen.
`
`-..___
`
`. .
`
`.
`
`180
`
`240
`
`300
`
`360
`
`
`
`of<f:>, o/J angles indicates the predominant
`
`0
`
`/J.
`/ lo
`[ I I I I 'l
`I I ' ..... 1
`
`.
`
`•
`
`_,,,, .
`
`••
`
`
`
`•
`
`antiparallel P-pleated
`
`(upper).
`FIG. 2 (lower).
`groups of Fab New after five cycles of real space refinement super­
`F1G. 1
`isomorphous replacement 2 A resolution
`• ., ..
`360r-��-.�.�,�.--.-� ....... -,�� --.� �� � ��� ...... �� ......
`.. . .. , .. . :
`. . . \
`360
`0
`-�� ... _ o:
`I
`.
`•": • I
`• •.•.
`. . .. �.,t: ·: .
`300
`'
`300
`-4\� • •• • I
`<t- ••
`/1 f • I
`• •'1� I
`• • • 1 e
`• •• • • I
`>'
`/
`0 ·� /,,,,,-•
`. , f • I I I
`I
`• I I I
`I I , I I I ( I
`2.40
`240
`I •
`'
`I
`_ _ 1 ,----
`'
`1 _____
`,_,
`'-------··
`.
`180
`180
`�
`0 /--- .-,
`I
`/
`f
`/ •• I
`/ . .,
`--- __ J
`-----
`I
`' '• .,
`I
`120
`120
`60
`60
`0 --.---- ----,,
`o�-----�---_-� .....
`� --·� ---�'"..-��-"���
`...... � �� ..... �� ........
`0
`60
`120
`0
`60
`120
`240
`180
`300
`360
`FJC. 3. Ramachandran plots of the VL and Q homology subunits ofFab New. The distribution
`
`sheet structure of the subunits. Glycine residues are indicated by 0.
`units (see Fig. 3) show a distribution
`structure present in V H• V L• CHl, and c ... As observed in
`the </>, tfJ angles for glycine
`
`
`
`of <f>, l/J angles which
`residues are scattered in the plot, frequently appearing in
`
`
`corresponds with the predominant antiparallel ,8-pleated sheet
`
`nonallowed regions for L-amino acids. Several other residues
`
`
`which occur outside areas of allowed conformation are found
`
`in bends of the polypeptide chains; it is expected that further
`
`several other protem structures,
`
`3 of 14
`
`BI Exhibit 1083
`
`

`

`588
`
`Structural Refinement of Fab New
`
`refinement will improve the angular values observed for these
`residues.
`
`
`
`Structural Comparison of Homology Subunits
`
`The structural homology of V H• VL, C81, and CL has been
`
`Initial matrices relating the Ca coordinates of the homology
`subunits were obtained from a small number of structurally
`
`equivalences new matrices were calculated and the process
`was iterated until no changes in equivalences were observed.
`A summary of the results is presented in Table Ill which lists
`
`subunits which were superimposed and compared by this
`process. The average value of the minimum base change
`
`lent amino acids is also given in Table lll.
`As can be seen in Table Ill, there is an even closer structural
`homology between VH, VL, C81, and CL in Fab New than that
`
`higher resolution of the Fab New model. Presumably the Ca
`
`(inverse! correlation between the number of Cas that are
`structurally equivalent and the average minimum base
`change per codon. Furthermore, when a restrictive condition
`
`average base change per codon becomes smaller, reflecting a
`
`alignment procedure described above. However, this proce·
`dure leads to amino acid sequence alignments that clearly
`
`(see Figs. 4 and 5). The closest sequence similarity in Fab
`
`described before (3, 4). A quantitative analysis of this homol­
`ogy using the method of Rao and Rossmann (9) is presented
`here. A $imil!lr !ln'1lysis h!l$ been m!lde by Rich<irdson et r;rl.
`the murine Fab McPC 603 fragment.
`(10) comparing the structures of superoxide dismutase and
`equivalent amino acids. The num.ber of equivalences was
`peptide chain for which the distances between putatively
`then extended by an automatic search for stretches of poly­
`equivalent Cas was smaller than 3.8 A. Based on the extended
`the number of Cas occurring at distances of less than 1.5 A
`and less than 3.0 A, for all the six possible pairings of
`necessary to exchange the codons of the structurally equiva­
`observed for McPC 603 Fab (10), probably reflecting the
`distances given in Table IIJ could become smaller with further
`impose with distances shorter than 1.5 A and 3.0 A is larger
`crystallographic refinement. The number of Cas which super­
`when comparing V H to V L and CHl to CL. Also, there is good
`for structural equivalence is imposed (dc"-<.A 5 1.5 A) the
`higher degree of conservation of amino acid sequences.
`It should be emphasized here that amino acid sequence
`information is not used in the quantitative three-dimensional
`Vi.., and between the Cii and Ct regions of immunoglobulins
`reflect the well established homologies between the V8 and
`New occurs between �l(y) and Ct(X), although, as shown in
`to that between � 1 and Ci. (see Figs. 6, 7, and 8). In addition,
`Table III, the structural similarity between V8 and v .. is close
`TABLE Ill
`Alignment of a-carbon coordinates of four homology subu nics of Fab
`(New) using method of Rao and Rossmann (9)
`base change
`Average
`Average
`Number of
`Number of
`minimum
`minimum
`c. pairs
`c. pairs
`base change
`dc.-ca{' I. 5 dc.-eoA'5. 1. 5 deo.,,.A s. 3. o dc.-eoAs. 3.o
`equiva·
`equiva·
`lenced with per codon for
`lenced with per codon for
`56
`81
`0.97
`0.98
`60
`82
`0.80
`0.71
`1.03
`66
`1.23
`40
`59
`1.04
`1.28
`1.24
`58
`1.04
`29
`25
`J.29
`1.40
`49
`27
`
`Subunits
`
`VwV1.
`C11i-CL
`c .. -v ..
`c .. -vH
`CHI-VI.
`c .. 1-v11
`
`Table III shows that there is considerable homology between
`
`regions of immunoglobulins.
`
`the V and C regions. These results can be interpreted to
`amino acid residues with highly preserved three-dimensional
`indicate that all the homology regions contain a basic core of
`structure. The chemical nature of these residues is also
`findings strongly support the postulate (15) of a gene duplica­
`preserved as indicated by the correspondingly lower values of
`the average base change per codon. As stated before (3) these
`tion mecha11i:;rn whida gave ri:;e w thi:! different humulugy
`The Cas of the homologous sequences, -Phe-Gly-Gly-Gly­
`(99 t.o 102) in Vi, and -Trp-Gly-Gln-Gly-(107 t.o 110) in VH, can
`be closely superimposed as can Ca atoms immediately preced­
`ing and following those residues. This conserved conformation
`gives no evidence supporting the postulate (16) that the
`glycine residues could serve as a pivot to allow for optimal
`quences has been proposed (4) in terms ofintersubunit (Vu to
`contacts between an antibody and its ligands. An alternative
`explanation for these constant, homologous V11 and Vi, se­
`V1,, see below) and intrasubunit contacts.
`defined from the model. The sequence -Val-Ser-Ser- (115 to
`The limits between the V and C homology regions can be
`117, Fig. 4) which is shared by y and µ.human H chains marks
`polypeptide chain the sequence -Ala-Ser-Thr (118 to 120)
`(106 t.o 108) corresponds to the COOH terminus of Vi.. and the
`residues -Gln-Pro-Lys- (110 to 112) constitute the NH2 termi­
`of VL or as the beginning residue of Ct. By the structural
`alignment described here however, Arg 109 can be properly
`In agreement with the Gm(4-) serological specificity oflgG
`considered as the COOH terminus of Vi,.
`This residue, corresponding to the Gm(l 7) allotype provides a
`better fit with the Fourier map than an arginine residue
`IgG New (17) which has been verified
`tation is reinforcR.d hy thP. Gm(l +) ."-P.rolngic.sil spe.r.ifir.it.y of
`(which correlates with Gm(4)) at that position. This interpre­
`
`the COOH terminus of Vu, and following a sharp bend in the
`
`marks the NH2 terminus of�l. The sequence -Thr-Val-Leu­
`
`nus of Ct. Thus, in the three-dimensional model Arg 109
`(usually assigned to VL) could be considered either as the end
`
`New (17), a lysine residue is placed at position 214 in Cu 1.
`
`analysis (12).
`
`by amino acid sequence
`
`Quaternary Structure
`
`Contacts between Homology Subunits -The closer contacts
`between the homology subunits of Fab New are diagrammat­
`
`indicates that the interactions between Vu and VL and be­
`tween � 1 and C.. are more extensive than those between Vu
`
`ically represented in Fig. 9 by lines joining Ca atoms sepa­
`rated by a distance of 8 A or less. This figure provides a
`description of regions of VH, Vi., � 1, and C.. in which there
`are higher density of contacts. Inspection of Figs. 8 and 9
`and C,,, 1 and those between Vi.. and Q. The fact that the V11
`and Ci.i 1 subunits (whose major axes make an angle smaller
`than 90°) interact more extensively than V1, and Ct (whose
`major axes makes an angle larger than 90°) is also reflected in
`Figs. 8 and 9.
`atom� situated at a distance not larger than 1.2 times their
`Intersubunit contacts between side chain and main chain
`contacting residues and the number of close contacts that
`
`van der Waals radii are given in Table IV. This table lists
`
`atoms from a given residue make with atoms of other residues.
`Evidently, amino acids with larger side chains have a poten­
`tial to make more contacts with other amino acids, thus for
`
`example, VH Trp 107 makes 29 intersubunit contacts, Trp 47
`
`4 of 14
`
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`

`

`Structural Refinement of Fab New
`
`589
`
`110
`
`120
`
`uo
`
`140
`
`150
`
`QPIAAPSVTLPPPSSllLQA•IATLVCLISDPYPGAV-TVAWl--ADal--
`
`120
`
`130
`
`140
`
`150
`
`---------------
`
`160
`
`ASTKGPSVFPLAPSSKSTSGGT�ALG
`
`CLVKDYPPSPV-TVSWW
`
`---SG••-•
`
`--------------
`
`61
`
`· · ·- - - rsvs1sa-
`
`70
`
`TPLaS•VTMLVWT-s
`
`- - - -- ----SSATLAITGLQAIDIADYYCQSYDl8L
`
`70
`
`90
`
`90
`
`100
`
`100
`
`l• • -vrGGGTILTVLl
`
`110
`
`109
`117
`
`90
`
`80
`-------K•OPSLaLSSVT
`
`AADTAVYYCaawLIAG
`
`•CIDVWGQG8LVTV••
`
`Ftc. 4. Amino acid sequences of the V,,, C... VH, and C,,1 homology regions ofFab New aligned by comparison of their three-dimensional
`170
`180
`190
`200
`310
`:220
`sequence. See Ref. 11 for the VL and C,, sequences, Ref. 12 for VH, and Ref. 13 for C,. l. Abbreviations for amino acids are as given in Ref. 14.
`struct11res. - - - indicate gaps introduced to maximize alignment of the three-dimensional structures. * indicate deletions in the VL
`
`-ALTS--
`
`GVBTPPAVLQSSGLYSL
`
`S8VVTVPSS8LGT-QTYIC•v••••••T1-vo11-v1r1ac
`
`homology regions of Fab New. The
`
`Fm. 5. Diagram of hydrogen bond­
`atoms for the VL, c.., V,., and C,.l
`ing (broken lines) between main chain
`hydrogen-bonded clusters correspond
`to the tw0 P-sheet structures of each
`ipate in intrachain and interchain di­
`sulfide bonds are underlined.
`
`subunit. Cysteine residues that partic­
`
`makes 28 contacts and Arg 43 makes 24 contacts.
`The contacts between V11 and VL are of particular interest
`in view of the fact that different H and L immunoglobulin
`
`chains can form structurally viable pairs. Three types of Vu-
`
`Vi. contacts will be considered in this discussion: first, the
`by residues which are invariant or semi-invariant in V11 and
`
`contacts which are a t the core of the contacting region, made
`
`V1• sequences; second, the contacts made by invariant or semi-
`
`5 of 14
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`BI Exhibit 1083
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`

`

`590
`
`Structural Refinement of Fab New
`
`Flo. 6. Stereo pair drawings of the
`a carbon backbones of the VL (top) and
`V" (bottom} 11ubunit.5. The 11ubunit.5 are
`viewed here in similar orientations.
`
`Flo. 7. Stereo pair drawing
`of the a
`Clirbon backbones of the Q (top) and
`C,. l (bottom) subunits viewed in simi­
`lar orientations.
`
`6 of 14
`
`BI Exhibit 1083
`
`

`

`Structural Refinement of Fab New
`
`591
`
`Fie. 8. Stereo pair drawing of the- oc
`carbon backbone of Fab New.
`
`invariant residues with hypervariable residues; and finally,
`those made between hypervariable residues.
`The core of the Vu-VL contacting region can be described as
`
`determined by residues Val 37, Gin 39, Leu 45, 'fyr 94, and
`
`at these positions could be accommodated by small displace­
`ments of the hypervariable peptide loops. These "idiotypic"
`interactions are consequently more difficult to assess. How­
`ever, they could perhaps explain the preferred reassociation
`observed between complementary H and L chains derived
`from a single immunoglobulin molecule (20). Most of the
`contacts discussed above consist of van der Waals interactions
`between hydrophobic side chains. However, a few hydrogen
`
`to VL Tyr 90 and/or v •. Arg 95. Also, an ion pair is fonned
`bonds can be indicated: V11 Gin al.I to v .. Gin 37, and V11 Asn 98
`In the Fab New model the contacts between V11 and V1• are
`between V11 Arg 43 and V� Asp 84.
`
`very close (Table IV), giving rise to a compact dimer. No
`haptens or even solvent molecules can be accommodated
`between V11 and Vi. beyond the combining site, a situation
`which is different from that described for an L-chain dimer
`(21).
`
`are extensive. The core of the contact area between C.11 and
`C,. is defined by Ci 1 residues Leu 128, Ala 129, Gly 143, and
`
`Pro 121, Val 135, and Leu 137. These residues appear to be
`invariant or nearly invariant in the H and L sequences from
`
`As shown in Table IV the interactions between C.11 and C,.
`Leu 145 and the structurally homologous C.. residues Phe 120,
`such as C-,.il: Phe 126, Pro 127, Thr 139, Lys 147, Phe 170, Pro
`different animal species. Most of the other contact residues
`171, Val 173, Gin 175, Ser 181, Val 185, Lys 218, and C..: Thr
`118, Ser 123, Glu 125, Glu 126, Lys 131, Thr 133, Thr 164, Ser
`homologous residues in the immunoglobul in chains from dif­
`of contacts (20 contacts) they make with each other and with
`/3-pleated sheet is more conserved than the rest of the CA
`(or genealogic tree) of distorted evolutionary distances. This
`
`177, Tyr 179, Lys 206, are also invariant or replaced by
`
`ferent animal species. In the contact area the central location
`of C111 Leu 128 and C.. Pbe 120 is reflected in the large number
`
`many other residues (see Table IV). As pointed out by Novotny
`and Franek (22) the amino acid sequence of the four-stranded
`
`regions in different animal species, leading to a dendrogram
`
`pattern in both C,11 and C,. in order to preserve
`
`observation can be analyred in terms of the structural model
`presented here as follows. The four-stranded f3 sheets of C111
`and C.. contain side chains which make intrasubunit contacts
`and in particular, they contain all or nearly all of the contact
`residues between Ci I and C,, (discussed above). Evidently,
`mutational events leading to amino acid replacements at
`these positions would have to occur in a complementary
`tertiary and
`quaternary immunoglobulin structure, and consequently they
`would be expected to occur at a slower rate than mutations in
`other regions of Ci 1 and C, ..
`As can be seen in Table JV the region immediately preced­
`ing the interchain disulfide bond does not provide close con­
`tacts between C,11 and C, .. In addition, the two strands of
`
`contacts with each other (about 50% of those listed in Table
`
`region, are nearly parallel and stacked on each other. The
`
`different animal species. For example, Tyr 35, Gln 37, Pro 43,
`
`Gin 39, Tyr 94 (replaced by Phe in a very few cases), and Trp
`
`Trp 107 in Vn and by residues Tyr 35, Gin 37, Ala 42, Pro 43,
`Tyr 86, and Phe 99 in V1•• These residues are structurally
`homologous with the except.ion that VL Ala 42 has no clear
`4). These homologous V11 and Vi. residues make numerous
`correspondence in V1, due to a structural "insertion" (see Fig.
`IV) or with other, nonhypervariable residues. The rings ofTrp
`107 (V11) and Pro 43 (V1), at the center of the VL -V11 contacting
`contact residues listed above are invariant or are replaced by
`homologous residues in Vi. V< and X) and V11 sequences from
`and Phe 99 appear constant in human L chains (K or A), and
`107 (replaced by Phe or Tyr in a very few cases) appear nearly
`constant in human H chains. Ala 42, Tyr 86 in Vi. and Val 37
`Ser 42, Phe 86, and Ile 37. The invariant or nearly invariant
`in VH are more frequently replaced by homologous residues:
`tween Ci 1 and Ci_, see below) for the property of different H
`provides a structural basis (together with interactions be­
`cules (see References 18, 19, and in particular 20, for a recent
`A second type of contact listed in Table IV is made between
`
`nature of these residues of the main VwV1. contacting area
`
`and L chains to recombine into new immunoglobulin mole­
`
`review and experimental data on this topic).
`
`constant or nonhypervariable residues and hypervariable res­
`idues. For example, the side chain atoms of Vn Trp 47, a
`constant residue in human, mouse, guinea pig, and in most
`rabbit immunoglobulin sequences, make close contacts with
`Ser 93, Leu 94, and Arg 95 in the third hypervariable region of
`V1 •• However, a large number of these contacts involve the
`peptide chain atoms of the V1• residues. Replacements in the
`
`VL side chains will not necessarily alter the nature of these
`
`contacts. Similar contacts appear to be made by V1• Leu 45
`(invariant or semi-invariant in human L chains) with the
`peptide chain at VH hype.rvariable position 104. Contacts of
`
`chain atoms of the fourth hypervariable region ofV11 in chains
`of different length than V11 New.
`The third type of contact to be discussed here is that made
`between hypervariable residues, such as those made between
`
`this type could also be made from Vi. Tyr 35 to the peptide
`VH Asn 98 and V1• Arg 95. These contacts are more difficult to
`evaluate in general terms (a) because the location of some of
`sequence, and (b) because it is possible that in other immu­
`the residues involved might be changed by further refinement
`to a larger extent than those of most other residues in the
`
`noglobulins, replacements by different amino acid side chains
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`7 of 14
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`BI Exhibit 1083
`
`

`

`592
`
`VH
`
`ti
`
`10
`
`,,
`
`Structural Refinement of Fab New
`CL
`VH
`
`117
`
`120
`
`121
`
`122
`12•
`
`123
`
`1S2
`
`163
`
`15'
`195
`UN
`
`117
`
`176
`
`17&
`
`2 1 2
`
`213
`
`...
`'5
`..
`"
`..
`"
`
`51
`58
`eo
`
`1°'
`
`101
`102 103
`105
`1oe
`107
`IOI
`1119
`
`110
`
`21'
`
`CH1
`
`CL
`
`7•
`
`120 ·==r
`
`161'
`
`1•
`
`1•
`171
`
`172
`
`l.I
`1.0
`
`161
`
`152
`
`163
`
`CH1
`
`128
`
`127
`
`1211
`1211
`
`130
`
`131
`
`132
`
`133
`
`1311
`''°
`1'2
`1'3
`
`141
`
`170
`
`171
`
`172
`
`173
`
`11•
`175
`176
`
`211
`
`219
`
`220
`
`VL
`
`31
`39
`40
`
`79
`80
`12
`
`112
`
`113
`
`,,,
`
`Fie. 9. Intersubunit a carbon contacts at distances of 8 A or less. Contacts are indicated by lines joining the corresponding amino oc.id
`T'l'sidue numbers. Numbers on the lin£i; indicate the contact distance (in Angstroms). Note the extensive VL .vH and C..·C.1 l interactions.
`fide bond do not closely interact with the rest of C11 ll or C,,.
`mental flexibility residing around this part of the immuno­
`polypeptide chain that come together at the interchain disul­
`globulin structure and in the immediately adjacent hinge
`region of the H chain.
`This region can be described as having a loose conformation,
`structural features are in agreement with the notion of seg-
`The results of several experimental approaches (see Chapter II
`
`with a lower electron density in the Fourier map. These
`
`Hyperuariable Regions, ldiotypes, and Combining Site­
`
`8 of 14
`
`BI Exhibit 1083
`
`

`

`hypervariable residues in the amino acid sequences of H and
`
`of an antibody molecule partially overlap. X-ray crystallo­
`graphic analyses (2-4, 21, 24, 25) provided an unequivocal
`confirmation of these conclusions and three-dimensional
`models of different immunoglobulin molecules in which the
`
`defined.
`
`in Ref. 23 for a comprehensive review) strongly suggested that
`L chains, idiotypic determinants, and combining site residues
`structural bases of these operational concepts could be further
`The hypervariable regions of the H and L chains are located
`can be folded without a defined secondary structure (such as a
`on exposed bends of the polypeptide chains, in regions which
`