Crystal structure of the human Fab fragment Kol and its comparison with the intact Kol molecule

Gel electrophoresis and molecular sieve chromatography were used to compare 17 different human KI type Bence Jones proteins including 5 for which the amino acid sequence is known. Although electrophoresis in the presence of NaDodSO4 showed uniformity of covalent dimer and monomer molecular weights, Sephadex chromatography under nondissociating conditions showed that monomers eluted with different apparent molecular weights. These differences were attributed to heterogeneity in light chain self-association; dimerization constants of the 17 proteins, calculated from a computer simulation of their behavior upon gel filtration, ranged from <103 to >106 M-1. The variable region, more specifically the third hypervariable region, appears to be responsible for the variation in the dimerization constant. Association properties of light chains of known sequence suggest that the presence of an aromatic or hydrophobic residue at position 96 enhances dimer formation whereas a charged residue at that position results in light chains remaining stable monomers. The location of hypervariable residue 96 within the amino-terminal portion of the joining segment of the variable region suggests that the joining region may account for the variability of selfassociation of light chains and, moreover, that it has a function in determining the selective association of immunoglobulin polypeptide chains.Studies on the physical and chemical properties of Bence Jones proteins-the homogeneous counterparts of immunoglobulin light chains-have provided considerable information concerning the structural basis for the generation of antibody diversity (1). Sequence analysis of K and X type Bence Jones proteins has revealed that light chains possess a primary structure characterized by variation in sequence among the first 107 amino-terminal residues, the variable half of the light chain (VL), and by a relatively invariant sequence of the ;107 carboxy-terminal residues, the constant half (CL). The VL consists of framework and hypervariable regions. Four subgroups of K chains and five subgroups of X chains have been defined structurally and immunochemically by the framework sequence homologies. The three hypervariable segments (HV1, HV2, HV3) contribute to the antigen-binding site (2).The VL and CL are under separate genetic control; recent studies of murine Bence Jones proteins indicate that the VL is encoded by two gene segments. One, the V gene, determines the sequence of the first t95 amino-terminal residues; the second, the joining (J) gene, determines the sequence of -10-12 carboxyl-terminal VL residues (3-6). The joining (J) segment of the V region contains a portion of segment HV3; thus, it contributes directly to the sequente diversity of the antigenbinding site. Its other functions, if any, have not as yet been determined. Whereas X Bence Jones proteins and light chains exist primarily as disulfide-linked covalent dimers, K Bence Jones proteins and light chains occur more commonly in the form of noncovalent dimers, stable monomers, ...

supporting

confidence: 53%

Self-association of human immunoglobulin kappa I light chains: role of the third hypervariable region.

Stevens¹,

Westholm²,

Solomon³

et al. 1980

“…The three masses in the map have approximately the same average value of density, suggesting that in any case the masses C and C* are very similar (20). Protein crystals showing different cell-to-cell organization of the same proteins have been reported (21). In the case of our F1 ATPase crystals, such an occurrence would be facilitated by -the fact that one of the three masses of density appears to be facing the solvent and it participates in little or no intermolecular contacts.…”

Section: Discussionmentioning

confidence: 90%

Structure of the mitochondrial F1 ATPase at 9-A resolution.

Amzel¹,

McKinney²,

Narayanan³

et al. 1982

The soluble portion (F1 ATPase) of the mitochondrial ATP-synthesizing system is a multisubunit enzyme of molecular weight 380,000. It is composed of five different subunits, a, .3, y, 8, and e. The subunit stoichiometry is not known but there are strong suggestions that it is a333y8e. We have determined the three-dimensional structure of the F1 ATPase of rat liver mitochondria to 9-A resolution by using x-ray diffraction techniques. The molecule appears to be formed by two equivalent halves, each formed by three regions ofapproximately equal size. These regions form a distorted hexagonal or octahedral arrangement. None of the regions form closed symmetrical trimers in the complex. It is proposed that, if the subunit stoichiometry is a3p3y8e, the major subunits exist in at least two different environments in the complex. In this arrangement, the different copies of the major subunits are functionally not equivalent. This observation appears to offer a natural explanation of the complicated binding and labeling data of F1 ATPases.The main pathway for the production ofATP in living organisms involves the phosphorylation of ADP by inorganic phosphate. It is clear from thermodynamic considerations that the levels of cellular ATP needed to perform biochemical phosphorylations, adenylations, and active transport should be above those determined by the hydrolytic equilibrium ATP + H20 = ADP + Pi. The ATP-synthesizing systems utilize the energy provided by the oxidation of substrates or by the capture of light quanta for the production ofsuch ATP levels. Coupling between the energy-yielding processes and the phosphorylation reaction is considered to be effected by an electrochemical proton gradient established across a biological membrane separating two compartments (1). The ATP-synthesizing system is then, according to this view, a proton-translocating ATPase.ATP-synthesizing complexes of similar structure are found in mitochondria, chloroplasts, and bacteria (for review, see refs. 2-6). They are large membrane-bound proteins composed of three morphologically distinct parts: a membrane sector, a connecting stalk, and a head piece or F1 ATPase.In this paper, we report an x-ray crystallographic study ofthe F1 ATPase from rat liver mitochondria (7). This protein has a molecular weight of380,000 and is composed offive nonidentical subunits a, A3, y, 8, and e (8) whose molecular weights estimated from NaDodSO4 gels are a, 62,500; (3, 57,000; 'y, 36,000; 8, 12,000; and E, 7,500. The stain intensity ofthese subunits in NaDodSO4 gels is compatible with a subunit stoichiometry of a3f33y5E (8). However, the subunit stoichiometry of F1 ATPases is still an unsolved problem and stoichiometries of the type a2322y282E2 are also considered possible in this system (for review, see ref. 6). The work described here provides information about the quaternary structure of the F1 ATPase of rat liver and in addition addresses the problem of the subunit stoichiometry of the enzyme. Our structure determination was carried out using the ...

“…The crystal structures of three immunoglobulin Fabs have been reported (1)(2)(3). One of these, the mouse myeloma protein McPC603, belongs to a class of immunoglobulins that bind specifically to phosphocholine.…”

mentioning

confidence: 99%

Crystal structure of galactan-binding mouse immunoglobulin J539 Fab at 4.5-A resolution.

Navia¹,

Segal²,

Padlan³

et al. 1979

An electron-density map of the mouse galactan-binding immunoglobulin 1539 (IgA2,K) Fab has been calculated to a resolution of 4.5 A by the method of heavy atom isomorphous replacement with four derivatives. The map has been interpreted with the aid of a com uter program which systematically searched for the best fit between the electrondensity map and the known coordinates of individual immunoglobulin domains. The quaternary structure of J539 Fab at this resolution appears similar to that of another mouse immunoglobulin, IgA2,K Fab, McPC603. The model coordinates for J539 Fab should allow us to proceed directly to a high-resolution structure determination without further heavy atom isomorphous replacement.The crystal structures of three immunoglobulin Fabs have been reported (1-3). One of these, the mouse myeloma protein McPC603, belongs to a class of immunoglobulins that bind specifically to phosphocholine. In this paper, we present the results of.a structural analysis at 4.5-A resolution of a second mouse Fab, the galactan-binding immunoglobulin J539. A molecular model has been constructed with the aid of a computer search procedure used previously in the interpretation of the electron-density map of an intact immunoglobulin (4). The model allows us to describe the quaternary structural interactions between the constituent domains and domain pairs of the J539 Fab and reveals a general similarity to McPC603. MATERIALS AND METHODSThe immunoglobulin J539 [a mouse IgA2,K; anti-(1-',6)-/-D-galactan] was isolated and purified as described (5), and pepsin fragments were obtained by using a published procedure (6). Useful heavy atom derivatives were obtained by soaking J539 Fab crystals in stabilizing solutions with 0.25 mM K2Pt(CNS)6 for 2 weeks, 1 mM K2PtCl4 for 1 week, 0.3 mM K2HgI4 for 2 weeks, and 50 mM KI/1 mM chloramine-T (Eastman Kodak) for 1 week. The iodine-derivatized crystals were washed with fresh stabilizing solution after the soaking period. Unique sets of three-dimensional reflection data to 4.5-A spacings were collected from native and derivative crystals with a Picker FACS-1 diffractometer operated in the T-scan mode and using Cu Ka radiation. The various data sets were placed on the same relative scale, and pseudo temperature factors were applied to correct for differences in intensity fall-off. The K2Pt(CNS)6 derivative was analyzed first, and difference Patterson maps, readily interpretable, revealed one site of substitution (Fig. 1). After preliminary refinement with projection data only, the coordinates and occupancy of the K2Pt(CNS)6 site were used to determine the signs of the centric native protein reflections. These signs were used to examine the K2PtCI4 and K2HgI4 derivatives by difference Fourier analysis. The variables for these three heavy atom derivatives were then subjected to alternating cycles of least squares refinement and phase determination with the three-dimensional data. The resulting phasesThe publication costs of this article were defrayed in part by page charge paym...