Using Molecular Dynamics Simulations as an Aid in the Prediction of Domain Swapping of Computationally Designed Protein Variants

Abstract

In standard implementations of computational protein design, a positive-design approach is used to predict sequences that will be stable on a given backbone structure. Possible competing states are typically not considered, primarily because appropriate structural models are not available.One potential competing state, the domain-swapped dimer, is especially compelling because it is often nearly identical to its monomeric counterpart, differing by just a few mutations in a hinge region. Molecular dynamics (MD) simulations provide a computational method to sample different conformational states of a structure. Here, we tested whether MD simulations could be used as a post-design screening tool to identify sequence mutations leading to domain-swapped dimers. We hypothesized that a successful computationally-designed sequence would have backbone structure and dynamics characteristics similar to that of the input structure, and that in contrast, domain-swapped dimers would exhibit increased backbone flexibility and/or altered structure in the hinge-loop region to accommodate the large conformational change required for domain swapping. While attempting to engineer a homodimer from a 51 amino acid fragment of the monomeric protein engrailed homeodomain (ENH), we had instead generated a domainswapped dimer (ENH_DsD). MD simulations on these proteins showed increased MD simulation derived B factors in the hinge loop of the ENH_DsD domain-swapped dimer relative to monomeric ENH. Two point mutants of ENH_DsD designed to recover the monomeric fold were then tested with an MD simulation protocol. The MD simulations suggested that one of these mutants would adopt the target monomeric structure, which was subsequently confirmed by X-ray crystallography.

Computational de novo design of antibodies binding to a peptide with high affinity

“…Due to the similarity of general structures of the antibodies, OptMAVEn mediated de novo antibody design could efficiently sample both local and non‐local contacts that are inherently present in the antibody structures using relatively large structural fragments (V, D, and J modular parts) extracted from known crystal structures, which is important for the successful design. All‐atom, explicit solvent MD simulations have previously proven to be effective at discerning active from inactive computationally designed Kemp eliminases (Kiss et al, ; Privett et al, ) and aiding in the prediction of domain swapping of computationally designed engrailed homeodomain protein variants (Mou et al, ). Inspired by their success, we carried out MD simulations for all de novo designed “germline” antibodies with the dodecapeptide prior to in silico affinity maturation and experimental validation.…”

“…We designed five de novo scFvs possessing distinct sequences compared to all existing natural antibody sequences that can bind with the dodecapeptide antigen using OptMAVEn. Since the stability and activity of a protein often depend not only on its static structure but also on its dynamic properties (Mou et al, ), an additional conformational sampling and stability evaluation using MD simulation was performed. The lack of sequence similarity between our designs and scFv‐2D10 demonstrates that the OptMAVEn procedure broadly samples the vast sequence space and arrives at designs unbiased by the original antibody residue composition.…”

Amino‐acid composition after loop deletion drives domain swapping

“…Hinge loops are likely to be strained in proteins that can domain‐swap . This conjecture is supported by the fact that the types of residues that occur in hinge loops in domain‐swapping proteins are different from the types of residues that occur in the loops of non‐domain‐swapping proteins . The intrinsic structural propensity of a residue determines whether it is locally stable in a given secondary structural element .…”

Computational protein design for given backbone: recent progresses in general method-related aspects

“…Mou et al used molecular dynamics simulation to investigate possible causes for a designed protein intended to form a homodimer turned out to be a domain-swapped dimer [43 ]. In simulations of the monomer, they found increased backbone flexibility and altered structures in a hinge loop involved in domain swapping.…”