ßγ-Crystallination Endows a Novel Bacterial Glycoside Hydrolase 64 with Ca2+-Dependent Activity Modulation.

The prokaryotic ßγ-crystallins are a large group of uncharacterized domains with Ca2+-binding motifs. We have observed that a vast number of these domains are found appended to other domains, in particular, the carbohydrate-active enzyme (CAZy) domains. To elucidate the functional significance of these prospective Ca2+ sensors in bacteria and this widespread domain association, we have studied one typical example from Clostridium beijerinckii, a bacterium known for its ability to produce acetone, butanol, and ethanol through fermentation of several carbohydrates. This novel glycoside hydrolase of family 64 (GH64), which we named glucanallin, is composed of a ßγ-crystallin domain, a GH64 domain, and a carbohydrate-binding module 56 (CBM56). The substrates of GH64, ß-1,3-glucans, are the targets for industrial biofuel production due to their plenitude. We have examined the Ca2+-binding properties of this protein, assayed its enzymatic activity, and analyzed the structural features of the ß-1,3-glucanase domain through its high-resolution crystal structure. The reaction products resulting from the enzyme reaction of glucanallin reinforce the mixed nature of GH64 enzymes, in contrast to the prevailing notion of them being an exotype. Upon disabling Ca2+ binding and comparing different domain combinations, we demonstrate that the ßγ-crystallin domain in glucanallin acts as a Ca2+ sensor and enhances the glycolytic activity of glucanallin through Ca2+ binding. We also compare the structural peculiarities of this new member of the GH64 family to two previously studied members.IMPORTANCE We have biochemically and structurally characterized a novel glucanase from the less studied GH64 family in a bacterium significant for fermentation of carbohydrates into biofuels. This enzyme displays a peculiar property of being distally modulated by Ca2+ via assistance from a neighboring ßγ-crystallin domain, likely through changes in the domain interface. In addition, this enzyme is found to be optimized for functioning in an acidic environment, which is in line with the possibility of its involvement in biofuel production. Multiple occurrences of a similar domain architecture suggest that such a "ßγ-crystallination"-mediated Ca2+ sensitivity may be widespread among bacterial proteins.

Mechanistic Insights from Replica Exchange Molecular Dynamics Simulations into Mutation Induced Disordered-to-Ordered Transition in Hahellin, a ßγ-Crystallin.

Intrinsically disordered proteins (IDPs) form a special category because they lack a unique well-folded 3D structure under physiological conditions. They play crucial role in cell signaling and regulatory functions and are responsible for several diseases. Although they are abundant in nature, only a small fraction of them have been characterized until date. Such proteins adopt a range of conformations and can undergo transformation from disordered-to-ordered state or vice versa upon binding to ligand. Insights of such conformational transition is perplexing in several cases. In the present study, we characterized disordered as well as ordered states and the interactions contributing the transitions through a mutational study by employing replica exchange molecular dynamics simulation with generalized Born implicit solvent model on a protein from the ßγ-crystallin superfamily. Most of the proteins within this superfamily are inherently ordered and highly stable. However, Hahellin, although a member of the ßγ-crystallin family, is intrinsically disordered in its apo-form which takes a well-ordered ßγ-crystallin fold upon binding to Ca2+. It is intriguing that the mutation at the fifth position of the canonical motif to Arg increases the domain stability in several ordered microbial ßγ-crystallins with concomitant loss in Ca2+ binding affinity. We carried out similar Ser to Arg mutations at fifth position of the canonical motif for the first time in an intrinsically disordered protein to understand the mechanistic insights of conformational transition. Our study revealed that newly formed ionic and hydrogen bonding interactions at the canonical Ca2+ binding sites play a crucial role in transforming the disordered conformation into ordered ßγ-crystallin.

Ca2+ and ßγ-crystallins: An affair that did not last?

BACKGROUND: During the last three decades, lens ß- and γ-crystallins have found a huge number of kin from numerous taxonomical sources. Most of these proteins from invertebrates and microbes have been demonstrated or predicted to bind Ca2+ involving a distinct double-clamp motif, which is largely degenerated in lens homologues. SCOPE OF REVIEW: The various aspects of transformation of ßγ-crystallins from a quintessential Ca2+-binding protein into a primarily structural molecule have been reviewed. MAJOR CONCLUSIONS: In lens members of ßγ-crystallins, the residues involved in Ca2+ binding have diverged considerably from the classical consensus with consequent reduction in their Ca2+-binding properties. This evolutionary change is congenial to their new role as robust constituents of lens. The exact functions of the residual affinity for Ca2+ are yet to be established. GENERAL SIGNIFICANCE: This review highlights the significance of reduction in Ca2+-binding ability of the ßγ-crystallins for lens physiology and why this residual affinity may be functionally important. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Microbial ßγ-crystallins.

ßγ-Crystallins have emerged as a superfamily of structurally homologous proteins with representatives across the domains of life. A major portion of this superfamily is constituted by members from microorganisms. This superfamily has also been recognized as a novel group of Ca(2+)-binding proteins with huge diversity. The ßγ domain shows variable properties in Ca(2+) binding, stability and association with other domains. The various members present a series of evolutionary adaptations culminating in great diversity in properties and functions. Most of the predicted ßγ-crystallins are yet to be characterized experimentally. In this review, we outline the distinctive features of microbial ßγ-crystallins and their position in the ßγ-crystallin superfamily.

Ca2+-binding motif of ßγ-crystallins.

ßγ-Crystallin-type double clamp (N/D)(N/D)XX(S/T)S motif is an established but sparsely investigated motif for Ca(2+) binding. A ßγ-crystallin domain is formed of two Greek key motifs, accommodating two Ca(2+)-binding sites. ßγ-Crystallins make a separate class of Ca(2+)-binding proteins (CaBP), apparently a major group of CaBP in bacteria. Paralleling the diversity in ßγ-crystallin domains, these motifs also show great diversity, both in structure and in function. Although the expression of some of them has been associated with stress, virulence, and adhesion, the functional implications of Ca(2+) binding to ßγ-crystallins in mediating biological processes are yet to be elucidated.