4 research outputs found
Decoding the molecular design principles underlying Ca<sup>2+</sup> binding to βγ-crystallin motifs
Numerous proteins belonging to the recently expanded βγ-crystallin superfamily bind Ca<sup>2+</sup> at the double-clamp N/D-N/D-X<sub>1-</sub>X<sub>2-</sub>S/T-S motif. However, there have been no attempts to understand the intricacies involving Ca<sup>2+</sup> binding, such as the determinants of Ca<sup>2+</sup>-binding affinity and their contributions to gain in stability. This work is an in-depth analysis of understanding the modes and determinants of Ca<sup>2+</sup> 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 Ca<sup>2+</sup>-binding protein, and clostrillin, a moderate-affinity protein. We monitored the consequences of these modifications on Ca<sup>2+</sup> binding by isothermal titration calorimetry, thermal stability and conformational and crystal structure analyses. We demonstrate that Ca<sup>2+</sup> 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 Ca<sup>2+</sup>-binding affinity, highlighting the basis of diversity found in these domains. A subtle change in the first site has a greater influence on Ca<sup>2+</sup> 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 Ca<sup>2+</sup>-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 Ca<sup>2+</sup> 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