PFIZER EX. 1113
`Page 1
`
`

`

`Biochemistry
`
`®Copyright 1975 by the American Chemical Society
`
`Volume 14, Number 22
`
`November 4, 197 5
`
`L-Phenylalanine:tRNA Ligase of Escherichia coli KlO.
`A Rapid Kinetic Investigation of the Catalytic Reaction t
`
`Peter Bartmann, Till Hanke, and Eggehard Holler*
`
`ABSTRACT: The kinetics of the amino acid activation and
`the transfer of the amino acid to tRNA have been investi(cid:173)
`gated for L-phenylalanine:tRNA ligase of Escherichia coli
`K10 by stopped-flow and radioactive techniques. The rapid
`kinetics were followed by the observation of the displace(cid:173)
`ment of the fluorescent dye, 6-p-toluidinylnaphthalene-2-
`sulfonate from the binding site of L-phenylalanine under
`conditions where a single active site of the enzyme was in(cid:173)
`volved. The following results are of particular interest. ( 1)
`Equilibrium binding of L-phenylalanine and tRNAPhe indi(cid:173)
`cates in each case two sites of interaction with an approxi(cid:173)
`mately tenfold difference of the binding affinity. (2) Exper(cid:173)
`imental conditions of the kinetic investigation were chosen
`to favor reactions at the high affinity binding sites. Under·
`those conditions, the rate constants have been evaluated at
`1 mM magnesium to be in the range 12-25 sec- 1 for the
`activation reaction and 42-77 sec- 1 for the reverse, the
`variation of the values depending on those of the dissocia-
`
`tion constants used for computation. The rate constant for
`the transfer reaction is 0.05 sec- 1 and for the reverse 0.19
`sec- 1• The forward reaction is rate limiting for the overall
`reaction at single turnover and steady-state conditions. (3)
`All rate constants depend on the concentration of magne(cid:173)
`sium. Evidence is provided that the transfer occurs via a
`productive enzyme-tRNAPhe complex which is in a magne(cid:173)
`sium-dependent equilibrium with an unproductive complex,
`high magnesium favoring the former. The position of the
`tRNA-CCA end in the productive complex is such, that the
`fluorescent dye can be displaced by Phe-tRNAPhe. The
`thermodynamics of the overall reaction have been treated
`on the basis of the partial reactions. The free enthalpy of
`the completed reaction was calculated to be very close to
`zero. The significance of the adenylate intermediate is dis(cid:173)
`cussed with respect to the product inhibition expected on
`the basis of the tendency of tRNAPhe and L-phenylalanine
`to form tight complexes with the enzyme.
`
`Previous investigations have provided considerable evi(cid:173)
`dence that the aminoacylation of tRNA as catalyzed by L(cid:173)
`amino acid:tRNA ligases proceeds via an activation of the
`amino acid and a subsequent transfer of this moiety to
`tRNA (Kisselev and Favorova, 1974, and references there(cid:173)
`in). Despite many efforts to establish the kinetics of these
`enzymes, none of them has been investigated beyond the
`time limits of the classical radioactive techniques as there
`are the ATP-[ 32P]PPi exchange and 14C amino acid label(cid:173)
`ing, to provide a detailed knowledge of the overall kinetics
`at conditions which are optimal for catalysis. In the present
`work we have attempted to resolve the catalytic steps, in
`particular those of the transfer reaction by means of
`stopped-flow experiments. We have also made classical ra(cid:173)
`dioactive measurements under single turnover and steady(cid:173)
`state conditions. However, we have not been following the
`kinetics of substrate binding. In connection with previous
`
`t From the Fachbereich Biologie, Universitiit Regensburg, 84
`Regensburg, Federal Republic of Germany. Received March 28, 1975.
`This work was generously supported by a grant from the Deutsche For(cid:173)
`schungsgemeinschaft and is part of the thesis of P.B.
`
`results we have drawn conclusions about the catalysis by L(cid:173)
`phenylalanine:tRNA ligase (Escherichia coli) which may
`be also valid for amino acid:tRNA ligases in general.
`
`Materials and Methods
`L-Phenylalanine:tRNA ligase (specific activity 53,600
`nmol mg- 1 hr- 1) was prepared from E. coli K10 in the
`presence of phenylmethanesulfonyl fluoride as described by
`Hanke et a!. (1974). Unfractionated tRNA was obtained
`according to Zubay (1962) from E. coli K10. Chromatog(cid:173)
`raphy on benzoylated DEAE-cellulose (Gillam eta!., 1967)
`and RPC-5 columns (Pearson et al., 1971) was applied to
`obtain tRNAPhe having an amino acid acceptance of 1250
`pmol/ Az6o unit (measured at pH 7 in HzO). Enzyme activ(cid:173)
`ity and tRNA charging capacity were determined as de(cid:173)
`scribed by Kosakowski and Bock (1970). Protein concentra(cid:173)
`tions were measured according to Lowry et al. ( 1961) and
`Waddell (1965).
`[ 14C]Phe-tRNAPhe was prepared as described previously
`(Bartmann et iii., 1974). Uniformly labeled L-[ 14C]pheny(cid:173)
`lalanine with a specific radioactivity of 450 Cijmol was ob-
`
`BIOCHEMISTRY, VOL. 14, NO. 22, 1975 4777
`
`PFIZER EX. 1113
`Page 2
`
`

`

`REFINED STRUCTURE OF A BENCE-JONES PROTEIN
`
`Sipos, T., and Merkel, J. R. (1970), Biochemistry 9, 2766.
`Smith, R. L., and Shaw, E. (1969), J. Bioi. Chern. 244,
`4704.
`Solomon, I. (1955), Phys. Rev. 99, 559.
`Stroud, R. M., Kay, L. M., and Dickerson, R. E. (1971),
`Cold Springs Harbor Symp. Quant. Bioi. 36, 125.
`Stroud, R. M., Kay, L. M., and Dickerson, R. E. (1974), J.
`Mol. Bioi. 83, 185.
`Titani, K., Ericsson, L. H., Neurath, H., and Walsh, K. A.
`(1975), Biochemistry 14, 1358.
`Trowbridge, C. G., Krehbiel, A., and Laskowski, M.
`(1963), Biochemistry 2, 843.
`
`Valenzuela, P., and Bender, M. L. (1969), Proc. Nat/.
`Acad. Sci. U.S.A. 63, 1214.
`Valenzuela, P., and Bender, M. L. (1970), Biochemistry 9,
`2440.
`Van Geet, A. L., and Hume, D. N. (1965), Anal. Chern. 37,
`979.
`Villanueva, G. B., and Herskovits, T. T. (1971), Biochemis(cid:173)
`try 10, 4589.
`Walsh, K., and Neurath, H. (1964), Proc. Nat/. Acad. Sci.
`U.S.A. 52, 884.
`Yguerabide, J., Epstein, H. F., and Stryer, L. (1970), J.
`Mol. Bioi. 51, 573.
`
`The Molecular Structure of a Dimer Composed of the
`Variable Portions of the Bence-Jones Protein REI
`Refined at 2.0-A Resolution t
`
`Otto Epp,* Eaton E. Lattman,t Marianne Schiffer,§ Robert Huber, and Walter Palm#
`
`ABSTRACT: The structure of the variable portions of a
`K-type Bence-Jones protein REI forming a dimer has been
`determined by X-ray diffraction to a resolution of 2.0 A.
`The structure has been refined using a constrained crystal(cid:173)
`lographic refinement procedure. The final R value is 0.24
`for 15,000 significantly measured reflections; the estimated
`standard deviation of atomic positions is 0.09 A. A more
`objective assessment of the error in the atomic positions is
`possible by comparing the two independently refined mono- ·
`mers. The mean deviation of main-chain atoms of the two
`chains in internal segments is 0.22 A, of main-chain dihe(cid:173)
`dral angles 6.3° for these segments. The unrefined molecu(cid:173)
`lar structure of the VREI dimer has been published (Epp,
`0., Colman, P., Fehlhammer, H., Bode, W., Schiffer, M.,
`Huber, R., and Palm, W. (1974), Eur. J. Biochem. 45,
`513). Now a detailed analysis is presented in terms of hy(cid:173)
`drogen bonds and conformational angles. Secondary struc-
`
`tural elements (antiparallel f3 structure, reverse turns) are
`defined. A more precise atomic arrangement of the amino
`acid residues forming the contact region and the hapten
`binding site is given as well as the localization of solvent
`molecules. Two cis-pralines (Pro-8 and Pro-95) were de(cid:173)
`tected. The intrachain disulfide bridge (Cys-23-Cys-88) oc(cid:173)
`curs statistically in two alternative conformations. The
`structure suggests reasons for strong conservation of several
`amino acid residues. The knowledge of the refined molecu(cid:173)
`lar structure enables crystal structure analyses of related
`molecules to be made by Patterson search techniques. The
`calculated phases based on the refined structure are much
`improved compared to isomorphous phases. Therefore the
`effects of hapten binding on the molecular struCture can be
`analyzed by the difference Fourier technique with more re(cid:173)
`liability. Hapten binding studies have been started.
`
`Immunoglobulins are proteins with specific antibody ac(cid:173)
`tivity. There exist several classes. The IgG class of immuno(cid:173)
`globulins is composed of two light and two heavy chains.
`The Bence-lones proteins excreted by patients with multi(cid:173)
`ple myeloma into the urine have been shown to be free light
`chains. The Bence-Jones protein REI is a human immuno(cid:173)
`globulin light chain of K type. The purification, crystalliza-
`
`t From the Max-Planck-Institut fiir Biochemic, 8033 Martinsried
`bei Miinchen, West Germany, and Physikalisch-Chemisches Institut
`der Technischen Universiti:t, Miinchen. Received May 30, 1975. The
`financial assistance of the Deutsche Forschungsgemeinschaft and Son(cid:173)
`derforschungsbereich 51 is gratefully acknowledged.
`t Present address: Rosenstiel Institute, Brandeis University, Wal(cid:173)
`tham, Massachusetts 02154.
`§Was on leave from: Division of Biological and Medical Research,
`Argonne National Laboratory, Argonne, Illinois 60439.
`#Present address: Institut fiir Medizinische Biochemic der Univ(cid:173)
`ersitiit Graz, Austria.
`
`tion, and sequence analysis has been described (Palm, 1970;
`Palm and Hilschmann, 1973, 1975; Palm, 1974). The crys(cid:173)
`tal structure of a dimer composed of the variable portions of
`this Bence-Jones protein at a resolution of 2.8 A was re(cid:173)
`ported (Epp eta!., 1974). Data to a resolution of 2.0 A have
`now been collected and the structure has been refined by
`constrained crystallographic refinement. The aim was to get
`a detailed insight into the conformation of this molecule
`(main chain, side chains, and bound solvent) and to obtain a
`model sufficiently accurate for its use in Patterson search
`techniques to determine the crystal structures of related
`molecules (Fehlhammer eta!., 1975). As refined phases are
`considerably better than isomorphous phases (Watenpaugh
`eta!., 1973; Deisenhofer and Steigemann, 1975; Huber et
`a!., 1974), the quality of difference Fourier maps will be
`much improved; this will make it possible to determine the
`structure bound haptens and the subtle structural changes
`which might occur upon binding.
`
`B I 0 CHEMISTRY, V 0 L. 1 4, N 0. 2 2, 1 9 7 5 4943
`
`PFIZER EX. 1113
`Page 3
`
`

`

`EPP ET AL.
`
`(3) Structure-- Factor Calculation
`
`(W. Steigemann and T. A. Jones) atomic scattering factors: Forsyth
`and Wells constants (1959), an overall temperature factor was used
`throughout.
`
`(4) R Value Calculation
`
`IF e II
`
`R is defined as
`
`LIIF0 I
`LIF0 1
`IF 0 I, observed structure-factor amplitude
`1Fel, calculated structure-factor amplitude
`For this calculation, as well as for the Fourier calculations, reflec(cid:173)
`tions of extremely bad correlation were excluded. The condition for
`exclusion was:
`
`211F0 1- !Fell
`IIF0 I+ IF ell > 1.2
`About 250 reflections were excluded. The innermost reflections to
`6.8-A resolution were omitted from all calculations (941).
`
`(5) Fourier Synthesis
`
`Grid: 1.1 A X 1.1 A X 1.0 A at the beginning to a resolution of 2.4
`A, later 0.8 A X 0.8 A X 0.8 A. During the course of the refinement
`the Fourier coefficients were mostly of the type (21F0 1- !Pel)
`expae. Also some Fourier syntheses with coefficients (31F 0 I -
`21F e I) expae were used. Difference Fourier maps were calculated
`with coefficients (IF0 1- 1Fel) expae·ae, calculated phase.
`
`(6) Computer Time on a Siemens 4004/150 (Cycle Time 0.75 J.LSec)
`
`Structure-factor calculation for 15,000
`reflections and 1630 atoms
`Fourier synthesis 1.62 X 10 5 grid points
`Real-space refinement for both chains in
`the asymmetric unit
`
`13,500 sec
`
`4000 sec
`17,000 sec
`
`Table I: Options and Specifications of Various Programs.
`
`(1) Model Building (Diamond, 1966)
`
`Probe
`
`1
`2
`3
`4
`5
`
`Probe
`Length
`oa
`2
`5
`6
`6
`
`1
`1
`2
`5
`6
`
`Filter Constants
`
`c,
`
`0.5
`0.5
`0.1
`0.1
`0.0
`
`1.oa
`0.5
`0.1
`0.0
`0.0
`
`C2•
`_2a
`10
`10-;
`10-4
`10-2
`10-2
`
`lQ-4
`10-4
`10-4
`10-4
`10-4
`
`Variation of
`Dihedral
`Angles
`</J,VJ ;x
`</J,VJ,x,r
`</J,V; ,x,r
`</J,VJ ,x,r
`</J,VJ ,x,r
`
`Eigenshifts are permitted if either A> C,e 2 or A> C2Amax•
`where A are eigenvalues of the normal matri.x. e2 is_ the residual.
`Variation of the folds of prolines x' of arginine, and w was not
`permitted. The angular value of ·1 (N-C"'-C) was fixed to a
`value of 109.65° including model-building procedure 7. In
`model-building 7 cis-pro lines were introduced (Huber and
`Steigemann, 1974). ¢,V; are main-chain dihedral angles, x are
`side-chain dihedral angles. The angular values of main chain
`and side chains were used and included wherever they were
`known, even if o_n-'ly~cr_u_d_e-'lY_·~~~~-~~~~~~~-
`(2) Real-Space Refinement (Diamond, 1971, 1974)
`
`Zone length
`Margin width
`Fixed radius of all atoms (A)
`Relative weights of C: N:O:S
`Relative softness of dihedral angles
`VI ,¢,x'-x 4
`x'
`8 I,() 2 ,!J 3 (proline)
`e 1 ,!J 2 (cysteine)
`T (N-Ca-C)
`w (torsion angle Ci-1"'-Ci-1-Ni-C/"')
`
`7
`6;8b
`1.55; 1.5QC
`6:7:8:16
`
`100
`1
`0.1
`0
`0.1
`0.1d
`
`Refinement of Scale Factor (K) and Background Level (d) Only
`
`Filter ratio Amin/Amax forK, d refinement
`Filter ratio for rotational refinement Amin/Amax
`Isomorphous map
`from difference Fourier map 2 on
`. from difference Fourier map 4 on
`from difference Fourier map 6 on
`The value of 0.00004 resulted in a proportion:
`of -~hifts applied of about
`Filter ratio for translational refinement
`Amin/Amax
`The value of 0.01 resulted in a proportion of
`shifts applied of about
`
`O.Ql
`
`0.005
`0.0005
`0.00007
`0.00004
`
`70%
`
`0.01
`
`100%
`
`a These values were used in model-building procedures 8 and 9 for probe length and filter constants. Also the variation of the angular value
`of r was allowed, but not for Gly, Ser, and Thr. b After introduction of w. CNew value estimated with the data at 2.1-A resolution. dw has
`been introduced after difference Fourier map 4 (data to 2.3-A resolution, R factor value 0.29, B = 23 A 2 ).
`
`Experimental Procedure
`The V REI dimer crystallizes in the space group P6 1. The
`hexagonal unit cell parameters are a = b = 75.8 A, c =
`98.2 A, 'Y = 120°. The asymmetric unit contains one dimer
`molecule. Intensity data were collected to 2.0-A resolution
`on a modified Siemens AED diffractometer using 8-28
`scan, with focus-to-crystal and crystal-to-detector distances
`of 30 em each. The intensity profile of each reflection was
`scanned twice in steps of 1 j 100° ( 44 steps over the whole
`reflection). The counting time per step was set inversely
`proportional to the intensity at the peak of the reflection;
`the upper limit was set at 2.4 sec/step for reflections to a
`resolution of 2.5 A. The reflections from 2.5- to 2.0-A reso(cid:173)
`lution were measured with 6 sec/step. Background was
`counted on both sides. The whole data set had been collect(cid:173)
`ed by diffractometers. The reflections were collected in
`shells of sin 8 fA.. Film data obtained from screenless preces(cid:173)
`sion photographs which were evaluated using the method of
`
`Schwager eta!. (Schwager eta!., 1975) were included and
`used for scaling purposes. The data were corrected for ab(cid:173)
`sorption by an empirical method (Huber and Kopfmann,
`1969). The complete intensity data set included 42,464
`measurements (of which 5454 are film data), which were
`merged to 14,993 independent reflections (0.69 of the possi(cid:173)
`ble reflections to a resolution of 2.0 A). All reflections to a
`resolution of 2.5 A were used (10381), except the innermost
`reflections to a resolution of 6.8 A (941) which are strongly
`influenced by the solvent continuum. To a resolution of 2.5
`A, 0.99 of the possible reflections is measured. From 2.5- to
`2.0-A resolution, only the reflections which could be ob(cid:173)
`served above the 20" significance level determined from
`counting statistics (3671) were used (e.g., 0.34 of the possi(cid:173)
`ble reflections in that range).
`The neglect of the innermost reflections to a resolution of
`6.8 A does not influence Diamond's real-space refinement
`procedure. In the refinement of PTI (Deisenhofer and
`
`4944
`
`B I 0 CHEMISTRY, V 0 L.
`
`] 4, N 0. 2 2, 1 9 7 5
`
`PFIZER EX. 1113
`Page 4
`
`

`

`REFINED STRUCTURE OF A BENCE-JONES PROTEIN
`
`Steigemann, 1975) the neglect of the innermost reflections
`improved the refinement of the positions of solvent mole(cid:173)
`cules. We omitted these reflections from all calculations
`and did not consider them in the interpretation of the elec(cid:173)
`tron density map. The R value for all measurements, de(cid:173)
`fined as
`
`R = (~ ~ ((J)h- Ihj)1 ~~Nh(I)h 2r 12
`is 0.05 ( (J)h is the average intensity of the Nh measure(cid:173)
`ments, fhj are the individual measurements of a reflection
`h). The Rsym values for individual crystals lie between
`0.022 and 0.065; the average Rsym is 0.04.
`The crystal structure of REI has been refined by a con(cid:173)
`strained crystallographic refinement described in the refine(cid:173)
`ment of the crystal structure of the bovine pancreatic tryp(cid:173)
`sin inhibitor (PTI) (Deisenhofer and Steigemann, 1975)
`and in the refinement of the structure of the complex be(cid:173)
`tween bovine trypsin and PTI (Huber et al., 1974). This
`procedure involves cycles consisting of phase calculation
`using the current atomic model, Fourier synthesis using
`these calculated phases and the observed structure-factor
`amplitudes, and Diamond's real-space refinement (Dia(cid:173)
`mond, 1971, 1974). At various stages (stagnation of further
`refinement), difference Fourier syntheses are calculated to
`detect and correct gross errors in the model (such as incor(cid:173)
`rect orientation of main-chain amides or side chains) and to
`localize solvent molecules. The stagnation of the refinement
`is reached, if the R factor value (definition see Table I)
`does not decrease further. Incorrectly oriented main-chain
`amides are rotated by reading the correct position of the
`carbonyl oxygen from the difference map and subjecting
`the whole chain to a model-building procedure (Diamond,
`1966). Side-chain orientations are corrected by rotating
`around the appropriate dihedral angles. Some characteris(cid:173)
`tics of the model-building procedure and the real-space re-'
`finement procedure are outlined in Table I. During the re(cid:173)
`finement 8 model-building procedures and 11 difference
`Fourier syntheses were calculated. The Fourier syntheses in
`the automatic cycles between these difference Fourier maps
`were mostly of the type (2IFol -IFJ) exp ac; a few (3IFJ -
`21 F J) exp ac were also used. Such syntheses increase the
`density gradients at the atomic positions and the speed of
`convergence. Table II shows the statistics of the course of
`the real-space refinement procedure.
`
`Results and Discussion
`Description of the Refinement. The starting model had
`been obtained through extensive real-space refinement of
`the model into the isomorphous Fourier map at 2.8-A reso(cid:173)
`lution. The above electron density map had first been aver(cid:173)
`aged over the two independent molecules (Epp et al., 197 4).
`The starting R factor (defined in Table I, 4) was 0.48. Dur(cid:173)
`ing the subsequent course of the refinement the two mole(cid:173)
`cules in the asymmetric unit were refined independently.
`The coordinates of the second molecule were obtained by
`applying the known local symmetry. After several cycles the
`R factor decreased to a value of 0.39. At this stage, the first
`difference Fourier map was calculated. The coordinates
`were plotted onto the Fourier map to check the progress.
`Misplaced side chains and several incorrectly oriented
`main-chain amides were detected (Table II). Between suc(cid:173)
`ceeding difference Fourier maps three to five automatic re(cid:173)
`finement cycles were performed, depending on the progress
`of the refinement. The refinement was started with reflec-
`
`Table II: Specifications of Difference Fourier Maps.
`
`Corrections
`- - · - - - - - - - - -
`in Each Map
`Reso- Overall Atom
`Side Chains
`Difference
`Main-Chain Amides lution Temp Fact. Radius
`Map No. R Value Chain 1 Chain 2
`(A2)
`(A)
`(r>(A)
`1
`0.390
`2
`0.350
`0.304
`3
`
`2.5
`2.4
`2.4
`
`28
`25
`23
`
`1.55
`1.55
`1.55
`
`10
`7
`4
`6
`2
`3
`11 solvent molecules
`4
`2
`26 solvent molecules
`4
`3
`40 solvent molecules
`1
`1
`39 solvent molecules
`introduction of cis-
`pralines (Pro-8,
`Pro-95)
`1
`42 solvent molecules
`1
`1
`36 solvent molecules
`
`41 solvent molecules
`
`55 solvent molecules
`
`53 solvent molecules
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`0.294
`
`0.282
`
`0.270
`
`0.264
`
`0.250
`
`0.241
`
`0.241
`
`0.241
`
`2.3
`
`2.3
`
`2.2
`
`2.2
`
`2.2
`
`2.1
`
`2.1
`
`2.0
`
`23
`
`23
`
`23
`
`23
`
`23
`
`23
`
`23
`
`21
`
`1.55
`
`1.55
`
`1.55
`
`1.55
`
`1.55
`
`1.55
`
`1.50
`
`1.50
`
`tion data to 2.5-A resolution. During the course of the re(cid:173)
`finement further data were included to 2.0-A resolution. An
`overall temperature factor was used, which was recalculat(cid:173)
`ed several times by comparing IFJ and IFJ and the temper(cid:173)
`ature factor changed if necessary. An average atomic radius
`of 1.55 A was used for all atoms throughout the refinement
`until difference Fourier map 9. Afterwards a new value of
`1.50 A was estimated by a trial calculation of atomic radii
`refinement in the real-space refinement step. This value is
`directly related to and consistent with the observed overall
`temperature factor of 21 A2• By the inspection of the differ(cid:173)
`ence Fourier maps, solvent molecules were detected and
`used in the phase calculations. The course of the difference
`maps is shown in Table II. The starting model had to be
`corrected in several segments. A number of main-chain am(cid:173)
`ides and side chains had to be rotated. For the correction of
`main-chain amides, the presence of two identical molecules
`in the asymmetric unit provided a very useful cross-check.
`In difference Fourier map 6, the segments around Pro-8
`and Pro-95 could be corrected. The local distribution of
`maxima and minima in these two regions was very similar
`and consistent in the two independent molecules, and it sug(cid:173)
`gested the presence of cis-peptide groups (Huber and
`StCigemann, 1974). After the introduction of these cis-pro(cid:173)
`lines,the refinement proceeded and stopped finally at an R
`factor value of 0.24. At this stage, 53 solvent molecules had
`been identified.
`Descriptipn of the Electron Density Map. Figure 1a and
`b is stereo pictures of the electron density and the model fit
`at two amino acid residues (Gln-37 and Tyr-71) 1 in order to
`
`1 Amino acid residue numbers are those of the VREI sequence. The
`nomenclature recommended by IUPAC-IUB (1970) is used in this
`paper with additional definitions as given by Diamond ( !966, 1971,
`1974). The coordinates of the VREJ dimer are available upon request.
`They are also in the Protein Data Bank of Brookhaven National Labo(cid:173)
`ratory. Coordinates as well as stereo drawings are contained in R.
`Feldmann's Global Atlas of Protein Structure on Microfiche.
`
`B I 0 CHEMISTRY, V 0 L. I 4, N 0. 2 2, I 9 7 5 4945
`
`PFIZER EX. 1113
`Page 5
`
`

`

`EPP ET AL.
`
`FIGURE 1: Electron density and model fit at two amino acid residues (top, Gln-37; bottom, Tyr-71). Contours in these figures from 0.3 e/A3 in
`steps of 0.3 e/ AJ.
`
`demonstrate the quality of the final Fourier map. A typical
`electron density for carbon is 0.8 e/ A3 and for oxygen 0.9
`ejA3.
`The final difference Fourier map is clear and suggests
`that the molecular structure is correctly interpreted. There
`is still some positive and negative residual density. Because
`of the restricted rotations about the S-S bond, cystine can
`exist in two mirror-image forms (Beychok, 1967). In the
`VREI 2 monomer there is one intrachain disulfide bridge
`(Cys-23-Cys-88). Both conformations, differing predomi(cid:173)
`nantly in the position of 23 S'Y, occur statistically in about
`the same proportion in each monomer. This is shown in Fig(cid:173)
`. ure 2a and b where sections 27-34 of difference Fourier
`maps (Fo- Fe) based on both conformations are drawn for
`monomer 2. Figure 2a shows the final difference map.
`Figure 2b shows a difference map based on the alterna(cid:173)
`tive cystine conformation in the molecule. The difference
`between the two configurations is essentially expressed in
`the value of the side-chain dihedral angle x3 (rotation about
`the S-S bond). It differs by 162°. The final difference Fou(cid:173)
`rier map also shows two statistical positions for the side
`chain ofGln-100.
`Besides these there are about 40 negative peaks with
`height -0.20 ejA3, 20 with height -0.25 ejA3 , and a few
`with height -0.30 ej A 3 . About 15 residual density peaks
`are positive, the highest is +0.25 ej A3 . The positive peaks
`are near atomic positions, the negative features occur main(cid:173)
`ly at carbonyl oxygens, at the badly defined region of the
`N-terminus, at Gly-41, and at C' and Nl" of lysine side
`chains. The negative residual density at carbonyl oxygens
`has also been observed in the PTI-trypsin complex and
`suggests that main-chain vibration affects predominantly
`
`2 Abbreviations used are: Fab', antigen-binding fragment of immu(cid:173)
`noglobulins; V REI. variable part of the Bence-Jones protein REI; V L,
`variable part of light chain; Ll, L2, and L3 are the first, second, and
`third hypervariable regions of light chains; K, X, the two major types of
`light chains differentiated by their C-terminal amino acid sequences;.
`Dnp, 2,4-dinitrophenyl.
`
`4946
`
`B I 0 CHEMISTRY, V 0 L. 1 4, N 0. 2 2, 1 9 7 5
`
`the carbonyl oxygens. Six of the 53 water molecules found
`at the end of the refinement, and included in the calcula(cid:173)
`tions with full occupancy and a temperature factor of 21
`A2, lie in negative density of -0.25 ej A3 •
`Accuracy of the Structure; Comparison of the Two Mo(cid:173)
`nomers. A comparison of the dihedral angles of the two
`main chains shows that only at theN- and C-termini, which
`are badly defined, at some Gly residues (Gly-57, Gly-64),
`and in some external loops (12-18, 26-34 first hypervaria(cid:173)
`ble region, 40-44, 78-81, 93-97 part of the third hypervar(cid:173)
`iable region) the differences between the two chains are
`considerably larger than the mean deviation <11¢,1/;) of
`9.6°. There are particularly large discrepancies in 4> and if;
`at Ser-26, Gln-27, and Asp-28. We observed no significant
`deviation in dihedral angles of residues forming the mono(cid:173)
`mer-monomer contact across the local diad (see Figure 5).
`The segments deviating significantly from the local symme(cid:173)
`try face the solvent, and the reasons for these structural dif(cid:173)
`ferences are unclear. As the difference map is featureless in
`these regions (Gln-27 is visible only to C'Y in both chains)
`these structures appear to represent real alternative confor(cid:173)
`mations. The mean deviation excluding these segments is
`<11¢,1/;): 6.3°. The same result is reflected by a comparison
`of the main-chain atoms (N, C'", Ci3, C, 0) of internal seg(cid:173)
`ments of both monomers (segments 4-10, 19-25, 35-38,
`45-49,62-77, 84-89, and 97-102). The mean deviation <r)
`is 0.22 A compared with 0.47 A for all atoms (the mean de(cid:173)
`viation of the internal segments for PTI and PTI complexed
`with trypsin is 0.25 A, Huber et a!., 1974). The average
`shift of main-chain atoms from the starting model (identi(cid:173)
`cal monomers) to the final model was 0.79 A neglecting N(cid:173)
`and C-terminal segments. A comparison of side-chain dihe(cid:173)
`dral angles shows larger differences, especially at residues
`Ser, Thr, Gin, and Lys.
`An objective assessment of the error of 0.2 A, in the
`atomic coordinates of the final model, is possible by com(cid:173)
`paring the two chains forming the dimer. This estimate is
`an upper limit because there may be small structural alter-
`
`PFIZER EX. 1113
`Page 6
`
`

`

`REFINED STRUCTURE OF A BENCE-JONES PROTEIN
`
`.. ;:::> @s
`c
`- ~,
`
`.
`
`35 CZ2
`
`~
`/
`';
`
`21 C01/;J :~ :,~
`,_.
`
`o 6 NOE
`
`71 CA o
`
`2 2.1 C"---.
`22 O<Y" ~
`0
`23 N
`71 N
`
`8
`
`'
`',
`'--I
`
`27
`
`28
`
`0 99 0
`
`30
`
`o33 CO!
`
`?1 CB
`'\,
`?1 CG "o
`
`~ :>
`,,
`
`88 SG
`
`!_:...-
`
`5 CE
`
`'~-l
`~:~m I
`
`'
`~ _,.
`r'
`['-- .... C5()33CD2 I
`i
`0
`-
`I
`
`8co1
`0
`
`o 6 NOE
`
`,. ",...-"'
`\
`( -<...::.:.. ... _)
`
`! 99 c
`' Qo=
`
`31
`
`99 N
`9 8 c
`
`/
`
`33
`
`32
`
`~:N c
`
`6 CA
`
`,.
`
`,:··-;, 0
`
`\ ... -·~'~'
`23 0
`o--"
`23 c
`
`( J 22 c
`v22~ 23 N
`
`0
`?1 N
`
`o 6 CB
`
`6
`
`2 Q
`
`; -
`.. -.',
`'/-':I
`,_,,
`
`'
`
`"
`
`88 N
`
`88CA
`
`I
`0
`3~
`@)
`-~89 a
`
`I
`
`0
`88 CB
`
`6
`
`2?
`
`2 8
`
`31
`
`033 C01
`0
`
`":)
`
`o99 o
`
`30
`
`2 9
`
`,.. -·
`
`Jt~
`
`,--/;-;;:~~":
`l - ~--=~--
`
`23 CA
`0
`
`a ©;:
`
`I
`
`.·.·.~4!J
`
`3 3
`
`7 N /1
`6 c
`-,6 CA
`
`1
`
`',_ ,'
`
`32
`
`--,
`
`I
`
`I
`
`FIGURE2: Sections 27-34 of difference Fourier maps (F0 - Fe) based on one of both possible cystine (Cys-23-Cys-88) conformations, respectively
`(shown is monomer 2). Contours from 0.10 ejA3 in steps of0.05 ejA3: (-)positive,(---) negative residual densities. (a) Final difference map; (b)
`an intermediate state of the refinement. This explains the pigh residual density below Met~4 SD.
`
`ations on dimer formation.
`An estimate of the accuracy of the final atomic positions
`can also be deduced by Cruickshank's formulas using the
`residual and the curvature of the electron density at the
`atomic positions (Cruickshank, 1949). The meah height of
`carbonyl oxygens in the final Fourier map is 0.9 ej A3. This
`yields a curvature of -!.32 ej A5 (Stout' and Jensen, 1968;
`Watenpaugh eta!., 1973). The resulting standard deviation
`is u(x) = 0.09 A. This is comparable to the values obtained
`foi: PTI-trypsin complex (Huber eta!., 1974).
`Description of the Monomer. The VREI monomer has a
`sandwich-like structure. The polypeptide chain can be di(cid:173)
`vided into nine segments, which form two halves partly con(cid:173)
`sisting of anti-parallel {3 structure. These segments are con(cid:173)
`nected by reverse turns. The two sheets cover a hydrophobic
`interior containing several invariant or almost completely
`conserved amino apid residues in K-light chains (Epp et a!.,
`1974). The upper part consists of five strands, the bottom
`
`part of three strands. The N-terminal strand adds to the
`upper sheet in a parallel and to the lower sheet in an anti(cid:173)
`parallel fashion. The lower part is a rather regular anti-par~
`aile! {3-pleated sheet. An analysis of the conformational an(cid:173)
`gles of the participating amino acid ·residues (5-7, 19-24,
`63-65, and 71-7 5) yields mean values for ¢ and 1/; of -117
`and + 140°, respectively, with standard deviations of 15 and
`16°'
`'
`The ¢ and 1/; values for a regular anti-parallel {3 structure
`({3-poly(L~afanine)) are -139 and +135° (Arnott et al.;
`1967). The remaining_ residues of the lower molecular part
`are involved in reverse turns. In the upper part, a regular
`antiparallel {3-structure conformation is formed by two
`strands containing the residues 34-38 and 85-89. The anal(cid:173)
`ysis of the conformational angles yields mean values for ¢
`and 1/; of -116 and + 137° with standard deviations of 13
`and 11 °, respectively. The other three strands of that mo(cid:173)
`lecular part are attached in a more irregular {3 structure.
`
`B IO CHEMISTRY, V 0 L. 1 4, N 0. 2 2, 1 9 7 5 494 7
`
`PFIZER EX. 1113
`Page 7
`
`

`

`Chain 1 Chain 2
`d(A) d(A) <d>(A)
`3.1
`3.0
`2.9
`3.0
`3.0
`2.9
`3.1
`3.1
`3.0
`2.8
`2.7
`2.8
`3.0
`3.1
`3.1
`3.0
`3.0
`3.0
`2.5
`3.0
`2.9
`
`3.0
`2.9
`
`3.1
`2.9
`3.2
`3.0
`3.1
`3.0
`2.8
`2.9
`2.8
`2.9
`2.9
`:u
`
`3.1
`
`3.0
`2.9
`2.9
`2.9
`2.8
`3.0
`
`3.2
`2.8
`2.8
`2.9
`3.0
`3.0
`2.6
`2.9
`2.9
`2.8
`2.9
`2.9
`
`3.0
`'3.0
`2.8
`3.1
`
`3.0
`
`3.0
`2.9
`2.9
`2.9
`2.8
`2.9
`
`2.7
`2.8
`2.9
`3.0
`2.9
`2.6
`2.8
`2.9
`2.8
`2.8
`3.0
`
`3.0
`3.0
`2.9
`3.0
`
`3.1
`2.9
`3.1
`3.0
`3.0
`2.8
`
`Table III: Intramolecular Hydrogen Bonds.
`
`Main Chain
`Gln-24 C=O
`Gln-24
`NH
`Thr-22 C=O
`Thr-22 NH
`Lys-103 C=O
`Gln-105 NH
`Thr-107 NH
`Asp-17
`NH
`Leu-78 C=O
`Leu-78 NH
`Ile-75
`NH
`Ile-75
`C=O
`Phe-7 3 C=O
`Phe-73
`NH
`Tyr-71 C=O
`Tyr-71
`NH
`Thr-69 C=O
`Tyr-32 NH
`Gly-68 C=O
`Gly-68
`NH
`Ala-51
`NH
`Glrt-89 C=O
`Gln-89
`NH
`Ile-48
`C=O
`Leu-47 NH
`Tyr-87 C=O
`Tyr-87
`NH
`Lys-45 C=O
`Lys-45
`NH
`Thr-85 C=O
`Thr-85
`NH
`Lys-42 NH
`Asrt-5 3 C=O
`Asn-53 NH
`Ser-76
`NH
`Thr-74 NH
`Thr-74 C=O
`Thr-72 C=O
`Thr-72
`NH
`Leu-104 NH
`Thr-102 C==O
`Thr-102 NH
`Gly-99
`NH
`
`Main Chain
`Thr-5
`NH
`Thr-5
`c==o
`Ser-7
`NH
`Ser-7
`C=O
`Leu-11 NH
`Leu-11 C=O
`Ala-13 C=O
`Ser-14 C=O
`Gln-16 NH
`Ser-17 C=O
`Val-1