Simulated Mutagenesis of the Hypervariable Loops of a Llama VHH Domain for the Recovery of Canonical Conformations

In this work, wildtype and mutated hypervariable regions of an anti-hCG llama VHH antibody were simulated via a molecular dynamics replica exchange method (REM). Seven mutants were simulated with the goal of identifying structural determinants that return the noncanonical HI loop of the wildtype antibody to the type I canonical structure predicted by database methods formulated for conventional antibodies. Two cases with three point mutations yielded a stable type I HI structure. In addition, other mutants with fewer mutations showed evidence of such conformations. Overall, the mutagenesis results suggest a marked influence of interloop interactions on the attainment of canonical conformations for this antibody. On the methodological front, a novel REM scheme was developed to quickly screen diverse mutants based on their relative propensities for attaining favorable structures. This multimutant REM (MMREM) was used to successfully identify mutations that stabilize a canonical HI loop grafted on the llama antibody scaffold. The use of MMREM and REM for screening mutants and assessing structural stability may be useful in the rational design of antibody hypervariable loops.