Tertiary structure of an amyloid immunoglobulin light chain protein: a proposed model for amyloid fibril formation.

Abstract

An immunoglobulin light chain protein was isolated from the urine of an individual (BRE) with systemic amyloidosis. Complete amino acid sequence of the variable region of the light chain (VL) protein established it as a kappa I, which when compared with other kappa I amyloid associated proteins had unique residues, including Ile-34, Leu-40, and Tyr-71. To study the tertiary structure, BRE VL was expressed in Escherichia coli by using a PCR product amplified from the patient BRE's bone marrow DNA. The PCR product was ligated into pCZ11, a thermal-inducible replication vector. Recombinant BRE VL was isolated, purified to homogeneity, and crystallized by using ammonium sulfate as the precipitant. Two crystal forms were obtained. In crystal form I the BRE VL kappa domain crystallizes as a dimer with unit cell constants isomorphous to previously published kappa protein structures. Comparison with a nonamyloid VL kappa domain from patient REI, identified significant differences in position of residues in the hypervariable segments plus variations in framework region (FR) segments 40-46 (FR2) and 66-67 (FR3). In addition, positional differences can be seen along the two types of local diads, corresponding to the monomer-monomer and dimer-dimer interfaces. From the packing diagram, a model for the amyloid light chain (AL) fibril is proposed based on a pseudohexagonal spiral structure with a rise of approximately the width of two dimers per 360 degree turn. This spiral structure could be consistent with the dimensions of amyloid fibrils as determined by electron microscopy.

Bence Jones KWR protein structures determined by X-ray crystallography

“…However, the molecules in the P3 1 21 crystals were degraded exclusively in the constant domains, formed hollow double-helical structures based on pseudo-sixfold screw symmetry and exhibited a counterclockwise handedness. As described below, these features are consistent with the amyloidogenic BJ BRE fibril model, which is composed solely of variable domains (Schormann et al, 1995;Steinrauf et al, 1999).…”

“…An extended antiparallel -sheet joining two molecules is thereby created. A similar hydrogen-bonding network is also found in the amyloidogenic BJ BRE model (Schormann et al, 1995;Steinrauf et al, 1999). In the BJ BRE network, three hydrogen bonds form an extended -sheet involving a sequence corresponding to that in BJ KWR.…”

“…An amyloid-fibril model based on the crystallographic packing of the amyloidogenic BJ BRE has been proposed which was formulated based only on the variable domains of a I light-chain dimer (Schormann et al, 1995;Steinrauf et al, 1999). The crystals in this case were monoclinic, but the BJ BRE variable domains arranged themselves in the form of a pseudo-sixfold helix to form a hollow cylinder of 115 Å diameter, as described in Schormann et al (1995) and in Steinrauf et al (1999). Fig.…”

Structural Insights into the Role of Mutations in Amyloidogenesis

“…This region is also shifted in I AL protein BRE when compared with a non-amyloidogenic protein (27). Crystallization studies of other AL proteins, AL-12 and AL-103, have also shown the 40 -44 loop to be disordered.…”

Effect of single point mutations in a form of systemic amyloidosis

“…Two fibril models of BRE are available in the Protein Data Bank. In both structures (PDB-ID: 1BRE and 1QP1), 38,39 the chains are arranged in a pseudohexagonal shape that contains six dimers in a 1808 turn. The diameter is about 100 Å and is in agreement with electron microscopy measurements of amyloid fibrils.…”