Structural alterations within native amyloidogenic immunoglobulin light chains

Abstract

Amyloid diseases are characterized by the misfolding of a precursor protein that leads to amyloid fibril formation. Despite the fact that there are different precursors, some commonalities in the misfolding mechanism are thought to exist. In light chain amyloidosis (AL), the immunoglobulin light chain forms amyloid fibrils that deposit in the extracellular space of vital organs. AL proteins are thermodynamically destabilized compared to non-amyloidogenic proteins and some studies have linked this instability to increased fibril formation rates. Here we present the crystal structures of two highly homologous AL proteins, AL-12 and AL-103. This structural study shows that these proteins retain the canonical germ line dimer interface. We highlight important structural alterations in two loops flanking the dimer interface and correlate these results with the somatic mutations present in AL-12 and AL-103. We suggest that these alterations are informative structural features that are likely contributing to protein instability that leads to conformational changes involved in the initial events of amyloid formation.

Figure 1. VL structure

A) Topological Diagram of the protein structure for AL-12 and AL-103 adapted from Schiffer. The two β-sheets of the domains have been separated. Residues that point towards the core are circled. CDR segments are highlighted in yellow. The β-strands have been encased within the arrows and are connected to their respective loops. AL-12 mutations are shown in red, AL-103 mutations in green.

Figure 2

Sequence Alignment of AL-12, AL-103 and the germline sequence κI O18/O8. Protein sequence alignment of AL-12 and AL103 with their parent germline sequence κI O18/O8 highlights a diversity of somatic mutations within the framework regions. AL-12 has 8 mutations, 7 of which are non-conservative, whilst AL-103 has 4, all of which are non-conservative including a proline insertion after position 95, referred to as 95ProIns. As a comparison to our previous work, the sequence of AL-09 has been included for completeness. AL-09 has 7 mutations, 3 of which are non-conservative. The generally accepted VL domain topology according to Kabat is provided (http://www.kabatdatabase.com/index.html). Germline sequence determination done using VBase website, (http://vbase.mrc-cpe.cam.ac.uk/). Secondary structure is shown (s= β-strand).

Figure 3

Mutation locations for AL-12 and AL-103. Molecular model for the crystal structure of AL-12 and AL-103 are shown in A and B respectively. The mutational positions for each crystal structure are indicated in grey spheres and labeled on each molecular model. The model for both AL-12 and AL-103 depicts the expected β-sheet rich, greek-key motif, typical of the immunoglobulin fold. Part C shows the superposition of AL-12 (yellow) and the germline protein κI O18/O8 (blue) highlighting that AL-12 adopts the canonical germline dimer interface. This is also true for AL-103 (not shown). The red arrows indicate the proline-40 and the proline-95 loops.

Figure 4

Structural Perturbations in both AL-12 and AL-103 are subtle. Upon superposition of the AL-12 or AL-103 it can clearly be observed that there are structural deviations in the region of proline 95. There is significant disruption to the backbone conformation in AL-103 in order to accommodate the proline insertion. Previous publications have shown similar small scale structural changes as those seen in the model structures for both AL-12 and AL-103,. These deviations cause re-arrangements of the hydrogen bonding patterns within this loop region. Despite the high resolution of diffraction, completeness and redundancy for both models the poor density in this region is indicative of greater mobility of this loop.

Figure 5

RMS comparison via Cα backbone superposition reveals larger areas of structural variability and instability. Using RMSD to compare Cα deviations gives a visual aid to help represent the structural deviations observed upon superposition of AL-12 (top) and AL-103 (bottom) models with either κI O18/O8 (A, B) or AL-09 (C, D). The smaller difference scale seen in A and B, when compared to C and D show that both AL-12 and AL-103 share more structural similarities with κI O18/O8 than AL-09. A and C highlight the small structural deviations seen in the proline-40 region of the AL-12 model as well as those in the proline-95 region. B and D highlight the relatively larger structural deviation seen in AL-103 in order to compensate for the insertion. The result of the insertion seems to be contained within the proline-95 loop and there are few knock-on consequences into the surrounding strands. The disruption represented here by high RMSD leads to hydrogen bond re-arrangements within the loops. This is significant as this directs hydrogen bonding away from the dimer interface.

Figure 6

Electron density for CDR3 loop region of AL-12 and AL-103 Figure 5 shows the electron density map, scaled at 2.0 sigma, for the proline-95 region of both AL-12 and AL-103. Key features and mutations in this region are labeled. For AL-12, there are significant density breaks throughout the loop region. In an attempt to satisfy all F_o-F_c multiple conformations were modeled for residue 96. The carbonyl group of proline-95 is modeled in both trans- and cis- conformations. The electron density breaks for AL-103 are less clear, but can be seen around the proline insertion. This insertion is accommodated to some extent by Tyr-96 being recruited further into the loop region to re-enforce intra-loop hydrogen bonding.