The structure determination of the variable portion of the Bence-Jones protein Au

The structure of a -type Bence-Jones protein variable fragment Au has been determined by molecular replacement methods using the known structure of an other Bence-Jones variable fragment Rei (Epp et al., Eur. J. Biochem. 45, 513 (1974)). The crystallographic R factor is 0.31 for about 4000 significantly measured reflections between 6.8 to 2.5 å. The Au protein forms a dimer across a crystallographic two fold axis. The spatial relationship of the two monomers, the conformation of the backbones and of the internal residues is extremely similar to that found in Rei.Extract from Dissertation, München (1974).on leave from the Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois 60439, USA.     

On the isolation, characterisation, and crystallisation of a human Bence-Jones protein of kappa type

The isolation and purification of the human Bence-Jones protein Rei is described. Physico-chemical data are given characterising the protein. It is shown that the protein could be crystallised. It is intended to use the crystals for single crystal X-ray diffraction studies.     

Structure of a novel Bence-Jones protein (Rhe) fragment at 1.6 A resolution

The crystal structure of Rhe, a lambda-type Bence-Jones protein fragment, has been solved and refined to a resolution of 1.6 A. A model fragment consisting of the complete variable domain and the first three residues of the constant domain yields a crystallographic residual RF value of 0.149. The protein exists as a dimer both in solution and in the crystals. Although the "immunoglobulin fold" is generally preserved in the structure, there are significant differences in both the monomer conformation and in the mode of association of monomers into dimers, when compared to other known Bence-Jones proteins or Fab fragments. The variations in conformation within monomers are particularly significant as they involve non-hypervariable residues, which previously were believed to be part of a "structurally invariant" framework common to all immunoglobulin variable domains. The novel mode of dimerization is equally important, as it can result in combining site shapes and sizes unobtainable with the conventional mode of dimerization. A comparison of the structure with other variable domain dimers reveals further that the variations within monomers and between domains in the dimer are coupled. Some possible functional implications revealed by this coupling are greater variability, induced fitting of the combining site to better accommodate antigenic determinants, and a mechanism for relaying binding information from one end of the variable domain dimer to the other. In addition to providing the most accurate atomic parameters for an immunoglobulin domain yet obtained, the high resolution and extensive refinement resulted in identification of several tightly bound water molecules in key structural positions. These water molecules may be regarded as integral components of the protein. Other water molecules appear to be required to stabilize the novel conformation.     

Structure of a dimeric fragment related to the lambda-type Bence-Jones protein: a preliminary study

A Bence-Jones protein, Rhe, isolated from the urine of a myeloma patient, has been crystallized. Immunological tests indicate that Rhe contains the constant region of a lambda-type light chain. Gel filtration and sodium dodecyl sulfate gel electrophoresis show that Rhe consists of two subunits, each about half the size of the light chain. Preliminary X-ray crystallographic results and density measurements show that Rhe possesses crystallographic 2-fold symmetry, indicating that the two subunits of Rhe are identical. This is the first report of evidence suggesting the possible occurrence in Bence-Jones protein of dinners comprised of solely the constant half of the light chain. Heavy-atom derivatives, possibly useful for a crystal structure analysis, have been prepared.     

Novel arrangement of immunoglobulin variable domains: X-ray crystallographic analysis of the lambda-chain dimer Bence-Jones protein Loc

We have characterized and crystallized a human lambda I light-chain dimer, Bence-Jones protein Loc, which has variable (V) region antigenic determinants characteristic for the lambda I subgroup and constant (C) region determinants of the C lambda I gene Mcg. The crystal structure was determined to 3-A resolution; the R factor is 0.27. The angle formed by the twofold axes of the V and C domains, the "elbow bend", is 97 degrees, the smallest found so far for an antibody fragment. The antigen-binding site formed by the two V domains of the Loc light chain differs significantly from those of other immunoglobulin molecules (light-chain dimers and Fab fragments) for which X-ray crystallographic data are available. Whereas, in other antibody fragments, the V domains are related by a local twofold axis, a local twofold screw axis with a translational component of 3.5 A relates the V domains in protein Loc. In contrast to the classic antigen binding "pocket" formed by V domain interactions in the previously characterized antibody structures, the V region associations in protein Loc result in a central protrusion in the binding site, with grooves on two sides of the protrusion. The structure of protein Loc indicates that immunoglobulins are physically capable of forming a more diverse spectrum of antigen-binding sites than has been heretofore apparent. Moreover, the unusual protruding nature of the binding site may be analogous to structures required for some anti-idiotypic antibodies. Further, the complementarity-determining residues form parts of two independent grooves.(ABSTRACT TRUNCATED AT 250 WORDS)     

The primary structure of the Bence-Jones protein Kue. The amino acid sequence of the variable part of a human L-chain of the kappa-type (author's transl)]

The complete primary structure of the variable region of Bence-Jones protein Kue was elucitated with the aid of a few tryptic peptides, as well as one chymotryptic and one BNPS-scatol fragment. As a consequence of the evident homologies to other proteins this protein belongs to subgroup k/I. Protein Kue has some amino acid exchanges in certain positions in common with other proteins, probably giving rise to a new sub-subgroup. The constant region shows no amino acid exchanges in comparison with other human kappa L-chains. With valine covering position 191, protein Kue should be grouped per definition as an allotype Km (3).     

Comparison of mouse mammary tumor virus-specific DNA in inbred, wild and Asian mice, and in tumors and normal organs from inbred mice.

The crystal and molecular structure of the dimer of variable domains of the Bence-Jones protein ROY has been determined by Patterson function search procedures, using the known structure of the protein REI. The structure has been partially refined at 3.0 Å resolution to a crystallographic R-factor value of 0.33. One of the 18 residues differentiating ROY from REI is the substitution of Tyr96 for Leu96, a substitution which makes the combining site of the ROY dimer larger. Substantial movement of Tyr49 suggests that point substitutions in the hypervariable segments may affect the conformation of neighbouring residues in the antigen-combining site, possibly producing differences in specificity larger than might otherwise be expected.     

Three-dimensional structure of an Fv from a human IgM immunoglobulin.

An IgM(kappa) immunoglobulin from a patient (Pot) with Waldenstrom's macroglobulinemia was hydrolyzed with pepsin to release a fragment consisting of the 'variable' (V) domains of the light and heavy chains plus eight residue 'tails' from the 'constant' (C) domains. The crystal structure of this fragment was determined at 2.3 A resolution by molecular replacement and crystallographic refinement methods. When examined separately, the light chain component closely resembles another human kappa chain (Rei) in both the beta-pleated sheet regions and the 'hypervariable' loops. The conserved pleated sheets in the heavy chain are similar to those in the human Kol IgG1 protein, but the third hypervariable loop in particular is different from that in any immunoglobulin structure described to date. As in the Kol protein, this loop blocks the access to any internal active site along the light-heavy chain interface. Unlike the Kol protein, however, the loop does not protrude beyond the boundaries of a conventional antigen combining site. Instead, it forms a very compact structure, which fills almost all residual space between the domains. This is an example of one dominant complementarity-determining region (CDR) essentially negating the diversity possible with five other CDRs in the two chains. Ordered water molecules are associated with light chain constituents along the interface, but not with CDR3 of the heavy chain. In screening exercises the Pot IgM failed to bind a wide variety of peptides. Together, the results suggest that ligand binding can only occur on external surfaces of the protein. These surfaces carry a limited number of side chains usually assigned to CDRs in more typical antibodies.     

Interconversion of conformational isomers of light chains in the Mcg immunoglobulins.

Previous crystallographic studies in this laboratory demonstrated that immunoglobulin light chains with the same amino acid sequence can have at least two and probably three or more conformations, depending on whether the second member of an interacting pair is a light or heavy chain. If a heavy chain is not available in the assembly medium, a second light chain plays the structural role of the heavy chain in the formation of a dimer. In the present work, the lambda-type light chains were dissociated from the heavy chains of a serum IgG1 immunoglobulin from the patient Mcg and reassembled noncovalently into a dimer. The reassembly process was completed by allowing the penultimate half-cystine residues to form an interchain disulfide bond. The covalently linked dimer was compared with the Mcg urinary Bence-Jones dimer, for which an atomic model has been fitted to a 2.3-A electron density map. The assembled dimer and the native Bence-Jones protein were indistinguishable in their chromatographic and electrophoretic properties, as well as in their activity in the binding of bis(dinitrophenyl)lysine. These results indicate that the light chains can be converted into the two types of Bence-Jones conformational isomers. The procedure was also reversed: the two Bence-Jones isomers were dissociated and reassembled as the single type of isomer associating with each of two heavy chains in the IgG1 protein. The change in activity occurring when a light chain associates with a heavy chain instead of a second light chain is illustrated by the fact that the Mcg IgG1 immunoglobulin does not bind dis(dinitrophenyl)lysine in measurable amounts.     

Kinetics of dimerization of the bence-jones protein Au

The dimerization reactions of complete Bence-Jones protein Au (VC-Au) and of its variable fragment (V-Au) were compared in 0.2 M (ionic strength) sodium phosphate buffer, pH 6.8 at 20° C. The dimerization constant for VC-Au (6.6 × 10⁴ M^?1) was slightly smaller than a previously published value for the fragment (1.1 × 10⁵ M^?1). The reaction enthalpies were positive for both processes. Temperature jump experiments exhibited two kinetic phases. The relaxation time of the fast phase as well as its concentration dependence and amplitude were almost identical for VC-Au and V-Au. Only small differences were observed in the slow phase. These close similarities between the reactions of the two proteins demonstrate that dimerization occurs mainly via interactions between the variable domains and that the constant domains interfere very little. From the observation of two relaxation times it follows that the dimerization mechanism for both VC-Au and V-Au must include at least three reacting species. Mechanisms with an isomerization between monomers in two conformational states and a single dimer species are excluded by the data. Alternative mechanisms with a single monomeric species but isomerization between dimers give a rather unsatisfactory fit. A good fit can be obtained if it is assumed that both monomers and dimers can exist in two states. Rate constants of the association and dissociation steps are of the order of 10⁷ M^?1 s^?1 and 10² s^?1. Isomerization rate constants are in the range of 10 s^?1.     

The molecular structure of a dimer composed of the variable portions of the Bence-Jones protein REI refined at 2.0-A resolution.

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 crystallographic refinement procedure. The final R value is 0.24 for 15000 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 monomers. The mean deviation of main-chain atoms of the two chains in internal segments in 0.22 A, of main-chain dihedral angles 6.3 degrees for these segments. The unrefined molecular structure of the VREI dimer has been published (Epp, O., 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 hydrogen bonds and conformational angles. Secondary structural elements (antiparallel beta 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-prolines (Pro-8 and Pro-95) were detected. The intrachain disulfide bridge (Cys-23-Cys-88) occurs statistically in two alternative conformations. The structure suggests reasons for strong conservation of several amino acid residues. The knowledge of the refined molecular 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 reliability. Hapten binding studies have been started.     

Thermodynamic and hydrodynamic study of Bence-Jones proteins

Four Bence-Jones proteins were studied under physiological conditions (10 mM phosphate buffer solution (pH 7.0) and 100 mM NaCl) by the circular dichroism, fluorescence, and analytical centrifugation methods. Combined analysis of the optical melting curves for the proteins and their fragments demonstrated that the stability of VAD protein and its constant half was decreased as compared with the other Bence-Jones proteins. This was correlated with the ability of both the whole protein and its constant (but not variable) part to form amyloid fibrils. The data on the correlation of the decreased stability with an abnormal interaction of two constant C_L domains are reported.     

Crystal structure of Bence Jones protein rhe (3 A) and its unique domain-domain association.

The crystal structure of protein Rhe, a lambda type V_L dimer, has been determined at a resolution of 3 Å by the method of multiple isomorphous replacement supplemented with anomalous scattering data. A crystallographic sequence was assigned from an interpretation of the electron density map in an optical comparator and is compared with a chemically determined partial amino acid sequence. The monomeric unit of Rhe, as determined crystallographically, contains 113 amino acids, 110 belonging to the variable region and three belonging to the constant segment of a light chain. The single polypeptide chain constituting the monomer forms a nine-stranded β-barrel characteristic of V domains. The β-pleated sheet surrounds an ellipsoidally shaped interior core of approximately 10 Å × 15 Å × 25 Å in size. The monomers that are related by the crystallographic dyad are held together as dimers by interdomain hydrogen bonds and hydrophobic interactions. At one end of the dimer is an opening which is lined exclusively by residues from the hypervariable regions.A comparison of Rhe with Rei, a kappa type V_L dimer (Epp et al., 1975), revealed that monomers of Rhe and Rei dimerized differently. Their respective dyad and pseudodyad of dimerization are not the same, and this causes a variation in the overall steric arrangement of the hypervariable regions in the two cavities. In adition a dissimilarity was observed in the non-hypervariable segment linking the first and second hypervariable regions. This segment is in the form of a loop and it includes most of the residues participating in the interdomain interactions stabilizing dimer formation in both proteins and these loop positions differ by as much as 7 Å. Our results also show that there is a good correlation between the dissimilarity of the loop position and the difference in the domain-association. Our preliminary analysis indicates that the positions of the corresponding non-hypervariable loops in V domains may be determined in part by the residues in the hypervariable regions.Accordingly, the three-dimensional structure of Rhe suggests that this nonhypervariable loop in V_L and its counterpart in V_H may have an important biological function in antibody specificity and variability by virtue of their influence over the architecture of the complementarity site.     

Is the formation of AL-type amyloid promoted by structural peculiarities of immunoglobulin L-chains? Primary structure of an amyloidogenic lambda-L-chain (BJP-ZIM)

A Bence-Jones protein (Protein ZIM) was isolated from the urine of a patient with myeloma-associated amyloidosis. The amino-acid sequence of the variable region of the carboxymethylated protein was established by automatic stepwise degradation of the enzymatically deblocked protein and tryptic peptides thereof. The protein is of the lambda-type of human immunoglobulin L-chains and is closely homologous to subgroup I. In the course of the tryptic digestion a precipitate was formed which showed properties characteristic of amyloid, such as staining with Congo red and green birefringence in polarized light. High-performance liquid chromatography was applied to separate these peptides. The precipitate consists of two peptides which coincide with position 19-45 of the variable and 129-140 of the constant part, respectively. Possible implications of this finding are discussed in the context of amyloid formation after limited proteolytic digestion.     

A molecular dynamics study of the C-terminal fragment of the L7/L12 ribosomal protein

The crystallographic dimer of the C-terminal fragment (CTF) of the L7/L12 ribosomal protein has been subjected to molecular dynamics (MD) simulations. A 90 picosecond (ps) trajectory for the protein dimer, 19 water molecules and two counter ions has been calculated at constant temperature. Effects of intermolecular interactions on the structure and dynamics have been studied. The exact crystallographic symmetry is lost and the atomic fluctuations differ from one monomer to the other. The average MD structure is more stable than the X-ray one, as judged by accessible surface area and energy calculations. Crystal (non-dimeric) interactions have been simulated in another 40 ps trajectory by using harmonic restraints to represent intermolecular hydrogen bonds. The conformational changes with respect ot the X-ray structure are then virtually suppressed.The unrestrained dimer trajectory has been scanned for cooperative motions involving secondary structure elements. The intrinsic collective motions of the monomer are transmitted via intermolecular contacts to the dimer structure.The existence of a stable dimeric form of CTF, resembling the crystallographic one, has been documented. At the cost of fairly small energy expenditure the dimer has considerable conformational flexibility. This flexibility may endow the dimer with some functional potential as an energy transducer.     

Immunoglobulin λ-chains. The complete amino acid sequence of a Bence-Jones protein

The total amino acid sequence of a lambda Bence-Jones protein has been established. The protein contains 211 residues, which include two methionine residues. Splitting with cyanogen bromide gave three fragments, the largest of which included the C-terminal half, which is common to other Bence-Jones proteins of the same type. The peptides obtained by tryptic, chymotryptic and peptic digestion were isolated and purified by paper-electrophoretic and chromatographic techniques. Reduction followed by carboxymethylation of the cysteine residues with radioactive iodoacetate was found to be a powerful tool in the isolation of some insoluble peptides. Unusual features of the molecule are the fact that it contains six cysteine residues and not five as observed in both kappa and lambda Bence-Jones proteins studied previously, and its size, which seems two residues smaller than the smallest Bence-Jones protein studied hitherto. The similarities and differences between this and other Bence-Jones proteins are discussed.     

Comparison of the three-dimensional structures of a human Bence-Jones dimer crystallized on Earth and aboard US Space Shuttle Mission STS-95

Crystals of a human (Sea) Bence-Jones dimer were produced in a capillary by vapor diffusion under microgravity conditions in the 9 day US Space Shuttle Mission STS-95. In comparison to ground-based experiments, nucleation was facile and spontaneous in space. Appearance of a very large (8 x 1.6 x 1.0 mm) crystal in a short time period is a strong endorsement for the use of microgravity to produce crystals sufficiently large for neutron diffraction studies. The Sea dimer crystallized in the orthorhombic space group P2(1)2(1)2(1), with a = 48.9 A, b = 85.2 A, and c = 114.0 A. The crystals grown in microgravity exhibited significantly lower mosaicities than those of ground-based crystals and the X-ray diffraction data had a lower overall B factor. Three-dimensional structures determined by X-ray analysis at two temperatures (100 and 293 K) were indistinguishable from those obtained from ground-based crystals. However, both the crystallographic R factor and the free R factor were slightly lower in the models derived from crystals produced in microgravity. The major difference between the two crystal growth systems is a lack of convection and sedimentation in a microgravity environment. This environment resulted in the growth of much larger, higher-quality crystals of the Sea Bence-Jones protein. Structurally, heretofore unrecognized grooves on the external surfaces of the Sea and other immunoglobulin-derived fragments are regular features and may offer supplementary binding regions for super antigens and other elongated ligands in the bloodstream and perivascular tissues.     

The immunosuppressive activity of peptide fragments of vaccinia virus C10L protein and a hypothesis on the role of this protein in the viral invasion

Our previous studies revealed that the 143-148 fragment of interleukin-1 receptor antagonist (IL-1 Ra) molecule with a Val-Thr-Lys-Phe-Tyr-Phe (VTKFYF) sequence inhibits the interleukin-1 (IL-1) interaction with its cellular receptor. The Val-Thr-Arg-Phe-Tyr-Phe (VTRFYF) sequence of the 322-327 fragment of the C-terminal domain of vaccinia virus protein related to the C10L vaccinia gene shows a very high homology to the 143-148 IL-1 Ra fragment, suggesting a similar inhibitory activity. To test this suggestion, we investigated the inhibitory activity of a series of synthetic peptides derived from 316 to 327 fragment of C10L on the interaction of IL-1 with its receptor. We also tested the peptides for their influence on the humoral and cellular immune response. The results indicate that biological activities of the C10L fragments are similar to those obtained for respective fragments of IL-1 Ra. The C-terminal domain of C10L protein can be easily folded into spatial structure similar to the crystallographic one of IL-1 Ra. Based on the crystallographic structure of IL-1 Ra, we constructed a 3-D model of the C10L protein. According to the model, the Val(322)-Asn(328) sequence is localized on the surface of the molecule and, therefore, it may be involved in the interactions with receptors. Our results indicate that the C10L viral protein can play an important role in vaccinia virus evasion of the host immune system. It may consist in the blockade of IL-1 receptors by the C10L protein, a homologue of the IL-1 Ra.     

Structure of the C-terminal domain of the ribosomal protein L7/L12 from Escherichia coli at 1.7 A

The structure of a C-terminal fragment of the ribosomal protein L7/L12 from Escherichia coli has been refined using crystallographic data to 1.7 A resolution. The R-value is 17.4%. Six residues at the N terminus are too disordered in the structure to be localized. These residues are probably part of a hinge in the complete L7/L12 molecule. The possibility that a 2-fold crystallographic axis is a molecular 2-fold axis is discussed. A patch of invariant residues on the surface of the dimer is probably involved in functional interactions with elongation factors.     

Role of cis- and trans-interactions in manifestations of amyloidogenic properties of variable domains of Bence-Jones proteins TIM and LUS

Intact Bence-Jones proteins TIM and LUS under simulated physiological conditions (10 mM phosphate buffer, pH 7.0, 100 mM NaCl, 37°C) did not display amyloidogenic properties. However, their isolated variable domains exhibit these qualities in full measure. Therefore, both intact proteins and their variable domains were studied using a complex of physical methods (scanning microcalorimetry, analytical centrifugation, optics) that allowed us to assess the stability of their tertiary and quaternary structures. The experimentally obtained thermodynamic functions indicated that the stability of iso-lated variable domains of TIM and LUS was comparable to the stability of similar domains in amyloidogenic proteins described earlier. However, inside the whole protein their stability was comparable to the stability of V_L domains of ordinary Bence-Jones proteins. The decreased stability of the isolated variable domains of TIM and LUS was shown to be due both to weak interactions between a pair of variable domains (trans -interaction) and to a natural lack of interaction with the con-stant domains (cis-interaction).