Controlling quaternary structure assembly: subunit interface engineering and crystal structure of dual chain avidin

Oligomeric proteins often form multiple quaternary structures, which can complicate their functional applications. Prior research has shown that protein scaffolds like avidin can be engineered for stability and specificity. However, controlling the exact assembly of these structures remains a challenge. No prior work had resolved how to guide the quaternary assembly of dual chain avidin (dcAvd) using interface mutations. This gap motivated the current study to explore how specific amino acid changes could influence dcAvd's quaternary structure. The pseudotetrameric nature of dcAvd offers potential for biotechnological applications, but its assembly is not fully understood. Researchers have already demonstrated that circular permutation can alter protein folding pathways. Yet, the role of interface residues in stabilizing a single assembly state was unknown. This study addresses that uncertainty by introducing mutations to control dcAvd's quaternary structure.

The aim of this research was to control the quaternary structure assembly of dual chain avidin (dcAvd) through interface engineering. dcAvd is a fusion of two circularly permuted monomers that can form two distinct pseudotetrameric states. The study sought to determine whether specific mutations could guide dcAvd into a single, stable assembly. The researchers hypothesized that modifying residues at the monomer-monomer interface could influence oligomerization. They focused on the 1-3 subunit interface, where symmetry allows multiple assembly configurations. The goal was to identify mutations that could lock dcAvd into a single quaternary structure while preserving its biotin-binding function. This approach could facilitate the design of more stable and predictable protein scaffolds. The findings may provide insights into how to control oligomeric protein assembly in general.

The study employed site-directed mutagenesis to modify residues at the interface of dual chain avidin (dcAvd). Researchers introduced the mutation I117C into one circularly permuted domain and scanned residues along the 1-3 subunit interface of the other domain. They tested the effects of these mutations on dcAvd's quaternary structure assembly. The modified dcAvd variants were analyzed using biophysical techniques to assess stability and oligomerization state. The researchers also evaluated biotin-binding activity at different pH levels to confirm functionality. Crystallography was used to determine the atomic structure of the dcAvd-biotin complex at 1.95 Å resolution. The study compared the structural outcomes of different mutations, focusing on V115H and I117H. The results were interpreted in the context of dcAvd's pseudotetrameric symmetry and interface interactions.

The mutation V115H induced a single, disulfide-locked quaternary assembly of dcAvd, whereas I117H did not guide oligomerization despite stabilizing the protein. The modified dcAvd forms retained their pseudotetrameric structure at both high and low pH. Biotin-binding levels remained comparable to wild-type chicken avidin in these variants. The crystal structure of the dcAvd-biotin complex at 1.95 Å resolution confirmed the formation of a functional pseudotetramer. This structure revealed the molecular basis for dcAvd's unique properties. The V115H mutation created a stable interface that restricted alternative quaternary states. The I117H mutation, while stabilizing, failed to enforce a single assembly pathway. These findings demonstrate that specific interface residues can control quaternary structure assembly in dcAvd.

The study shows that interface engineering can guide quaternary structure assembly in dual chain avidin (dcAvd). The V115H mutation successfully locked dcAvd into a single, disulfide-bridged pseudotetrameric state. This modification did not compromise biotin-binding activity or stability across pH ranges. The crystal structure at 1.95 Å resolution confirmed the formation of a functional pseudotetramer. These findings suggest that residue-specific modifications can control oligomerization pathways in engineered proteins. The I117H mutation, while stabilizing, failed to enforce a single assembly state. This highlights the importance of residue selection in interface engineering. The results provide a framework for further engineering of dcAvd and other oligomeric proteins. The study supports the hypothesis that interface residues can be used to direct quaternary structure assembly.