HAD superfamily members share a Rossman‑like core domain and sometimes contain a cap module that regulates access of substrates to the active site. Insight into the co-variance of cap and core domains and thus HAD structure/function relationship is critically needed to guide target selection and assist computational docking. The HAD Bridging Project and Superfamily/Genome and Computation Cores combined efforts to carry out a quantitative analysis of over a hundred core-domain-only and cap-domain-only structures to assess the impact of cap insertion on divergence of the core. The results detailed in this publication revealed basic protein design principles that suggest intra-molecular coevolution in which the thermodynamically stable superfold diverges differentially in the context of an accessory domain. The relationships between sequence and structure for other enzyme superfamilies and families with multiple domains may be elucidated with a similar strategy, thereby helping in the development of functional assignments strategies that are broadly applicable.
Although the universe of protein structures is vast, these innumerable structures can be categorized into a finite number of folds. New functions commonly evolve by elaboration of existing scaffolds, for example, via domain insertions. Thus, understanding structural diversity of a protein fold evolving via domain insertions is a fundamental challenge. The haloalkanoic dehalogenase superfamily serves as an excellent model system wherein a variable cap domain accessorizes the ubiquitous Rossmann-fold core domain. Here, we determine the impact of the cap-domain insertion on the sequence and structure divergence of the core domain. Through quantitative analysis on a unique dataset of 154 core-domain-only and cap-domain-only structures, basic principles of their evolution have been uncovered. The relationship between sequence and structure divergence of the core domain is shown to be monotonic and independent of the corresponding type of domain insert, reflecting the robustness of the Rossmann fold to mutation. However, core domains with the same cap type share greater similarity at the sequence and structure levels, suggesting interplay between the cap and core domains. Notably, results reveal that the variance in structure maps to α-helices flanking the central β-sheet and not to the domain-domain interface. Collectively, these results hint at intramolecular coevolution where the fold diverges differentially in the context of an accessory domain, a feature that might also apply to other multidomain superfamilies.