Decoding the molecular design principles underlying Ca²⁺ binding to βγ-crystallin motifs

Numerous proteins belonging to the recently expanded βγ-crystallin superfamily bind Ca²⁺ at the double-clamp N/D-N/D-X_1-X_2-S/T-S motif. However, there have been no attempts to understand the intricacies involving Ca²⁺ binding, such as the determinants of Ca²⁺-binding affinity and their contributions to gain in stability. This work is an in-depth analysis of understanding the modes and determinants of Ca²⁺ binding to βγ-crystallin motifs. We have performed extensive naturally occurring substitutions from related proteins on the βγ-crystallin domains of flavollin, a low-affinity Ca²⁺-binding protein, and clostrillin, a moderate-affinity protein. We monitored the consequences of these modifications on Ca²⁺ binding by isothermal titration calorimetry, thermal stability and conformational and crystal structure analyses. We demonstrate that Ca²⁺ binding to the two sites of a βγ-domain is interdependent and that the presence of Arg at the fifth position disables a site. A change from Thr to Ser, or vice versa, influences Ca²⁺-binding affinity, highlighting the basis of diversity found in these domains. A subtle change in the first site has a greater influence on Ca²⁺ binding than a similar alteration in the second site. Thus, the second site is more variable in nature. Replacing an acidic or hydrophobic residue in a binding site alters the Ca²⁺-binding properties drastically. While it appears from their binding site sequence that these domains have evolved randomly, our examination illustrates the subtlety in the design of these modules. Decoding such design schemes would aid in our understanding of the functional themes underlying differential Ca²⁺ binding and in predicting these in emerging sequence information

Aggregation-Prone Near-Native Intermediate Formation during Unfolding of a Structurally Similar Nonlenticular βγ-Crystallin Domain

The folding and unfolding of structurally similar proteins belonging to a family have long been a focus of investigation of the structure–(un)­folding relationship. Such studies are yet to reach a consensus about whether structurally similar domains follow common or different unfolding pathways. Members of the βγ-crystallin superfamily, which consists of structurally similar proteins with limited sequence similarity from diverse life forms spanning microbes to mammals, form an appropriate model system for exploring this relationship further. We selected a new member, Crybg3_D3, the third βγ-crystallin domain of non-lens vertebrate protein Crybg3 from mouse brain. The crystal structure determined at 1.86 Å demonstrates that the βγ-crystallin domain of Crybg3 resembles more closely the lens βγ-crystallins than the microbial crystallins do. However, interestingly, this structural cousin follows a quite distinct (un)­folding pathway via formation of an intermediate state. The intermediate species is in a nativelike conformation with variation in flexibility and tends to form insoluble aggregates. The individual domains of lens βγ-crystallins (and microbial homologues) do not follow such an unfolding pattern. Thus, even the closest members of a subfamily within a superfamily do not necessarily follow similar unfolding paths, suggesting the divergence acquired by these domains, which could be observed only by unfolding. Additionally, this study provides insights into the modifications that this domain has undergone during its recruitment into the non-lens tissues in vertebrates

Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike protein in complex with ACE2 and 2 potent neutralizing antibodies

The recently reported “UK variant” (B.1.1.7) of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-electron microscopy (cryo-EM) structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent neutralizing antibody fragments.Medicine, Faculty ofNon UBCBiochemistry and Molecular Biology, Department ofReviewedFacultyResearcherPostdoctoralGraduat