Stabilizing an amyloidogenic λ6 light chain variable domain.

Light chain amyloidosis is a lethal disease where vital organs are damaged by the fibrillar aggregation of monoclonal light chains. λ6a is an immunoglobulin light chain encoded by the germ-line gene segment implicated in this disease. AR is a patient-derived germ-line variant with a markedly low thermodynamic stability and prone to form fibrils in vitro in less than an hour. Here, we sought to stabilize this domain by mutating some residues back to the germ-line sequence, and the most stabilizing mutations were the single-mutant AR-F21I and the double-mutant AR-F21/IV104L, both located in the hydrophobic core. While mutation Arg25Gly in 6aJL2 destabilized the domain, mutating Gly25 back to arginine in AR did not contribute to stabilization as expected. Crystallographic structures of AR and 6a-R25G were generated to explain this discrepancy. Finally, 6a-R25G crystals revealed an octameric assembly which was emulated into 6aJL2 and AR crystals by replicating their structural parameters and suggesting a common assembly pattern. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession numbers 5IR3 and 5C9K.

Simple approach for ranking structure determining residues.

Mutating residues has been a common task in order to study structural properties of the protein of interest. Here, we propose and validate a simple method that allows the identification of structural determinants; i.e., residues essential for preservation of the stability of global structure, regardless of the protein topology. This method evaluates all of the residues in a 3D structure of a given globular protein by ranking them according to their connectivity and movement restrictions without topology constraints. Our results matched up with sequence-based predictors that look up for intrinsically disordered segments, suggesting that protein disorder can also be described with the proposed methodology.

Site-directed mutagenesis reveals regions implicated in the stability and fiber formation of human λ3r light chains.

Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this work, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40-60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. This mutagenic approach helped to identify key regions implicated in λ3 AL.