Pitfalls of Molecular Replacement: the Structure Determination of an Immunoglobulin Light-Chain Dimer

Abstract

The structure of protein Cle, a human light-chain dimer from the ,;AII subgroup, was determined using 2.6A data; the R value is 18.4%. The structure was solved, after a false start, by molecular replacement with the ;.II/V Mcg protein as a search structure. When the refinement did not proceed beyond an R value of 27 %, it was discovered that while the constant domains were in their correct positions in the unit cell, the incorrect variable domains were used for defining the molecule. The correct solution required a rotation of 180 around the local twofold axis that relates the two constant domains of the dimer. The correct variable domain positions overlap about 70% of the same volume as the incorrect ones of a symmetry-related molecule. The refinement distorted the geometries of the domains. Though the constant domains were in their correct positions, the r.m.s. (root-mean-square) deviation of the Co~ atom position was 1.2A when the two constant domains were compared. For the correct structure, this value is 0.5 A,. The q9 and ~p angles, the r.m.s, chiral value and the free R value, even when calculated a posteriori, were good indicators of the correctness of the structure. The quaternary structure of the Cle molecule is similar to that in Mcg (crystallized from ammonium sulfate); the elbow bend is 115. However, the arrangement of the variable domains differs from that observed in other variable domain dimers. The variable domains of Cle are 0.7 ,~, closer than in Mcg or variable dimer Rei. The hydrogen bonding at the interface of the two domains is novel. Residues Tyr36 from both monomers form a hydrogen bond that is part of a network with the Gin89 residues from both monomers. For the first time hydrogen bonds were observed between the main-chain peptide N and O atoms of the complementarity-determining region CDR2 and CDR3 segments of both monomers.

Role of domain interactions in the aggregation of full-length immunoglobulin light chains

“…This is readily noted by the similar positions of C L domain cross-peaks in the C215S JTO-FL and WT JTO-FL proteins, where in the latter case the C L -C L interface is completely formed (compare green and blue in Fig. 5A), as shown in the X-ray structure of the FL LC (10). Thus, the presence of the V L interface in the full protein stabilizes interactions between the C domains.…”

“…The mechanisms by which amyloidogenesis occurs and the processes leading to amyloid accumulation in organs are not well understood. X-ray structures of full-length (FL) Ig LCs show them to be dimeric, with each LC composed of N-and C-terminal variable (V L ) and constant (C L ) domains, respectively (10). Each LC monomer in the dimeric structure is arranged to form substantial V L -V L and C L -C L domain-domain interfaces, as shown schematically in Fig.…”

Heat-induced formation of a specific binding site for self-assembled congo red in the V domain of immunoglobulin L chain ?

“…Knowledge of the N‐terminal amino acid sequence made it possible to find the homologous protein of known crystal structure in the Protein Data Bank. The Loc Lλ chain was chosen as the model molecule for analysis 34–36. Figure 7 shows two versions of the L chain λ ( Loc ): one not altered by digestion, and its digested derivative with 20 N‐terminal amino acid residues removed.…”

Site-directed Mutagenesis Reveals Regions Implicated in the Stability and Fiber Formation of Human λ3r Light Chains

“…The integrated reflections were sorted, scaled, and truncated with SORTMTZ, SCALA, and TRUNCATE (37) from the CCP4 suite, respectively. Molecular replacement was carried out in PHASER (38) using the edited Protein Data Bank code 1LIL atomic coordinates, corresponding to the 3 immunoglobulin Cle, as the starting model (24). The resulting model for each protein was subjected to rigid body refinement followed by restrained refinement in REFMAC5 (39).…”