Maroor K. Jobby

The βγ-Crystallin Superfamily Contains a Universal Motif for Binding Calcium,

The betagamma-crystallin superfamily consists of evolutionarily related proteins with domain topology similar to lens beta- and gamma-crystallins, formed from duplicated Greek key motifs. Ca(2+) binding was found in a few betagamma-crystallin members earlier, although its prevalence and diversity as inherent molecular properties among members of the superfamily are not well studied. To increase our understanding of Ca(2+) binding in various betagamma-crystallins, we undertook comprehensive structural and Ca(2+)-binding studies of seven members of the superfamily from bacteria, archaea, and vertebrates, including determination of high-resolution crystal structures of three proteins. Our structural observations show that the determinants of Ca(2+) coordination remain conserved in the form of an N/D-N/D-#-I-S/T-S motif in all domains. However, binding of Ca(2+) elicits varied physicochemical responses, ranging from passive sequestration to active stabilization. The motif in this superfamily is modified in some members like lens crystallins where Ca(2+)-binding abilities are partly or completely compromised. We show that reduction or loss of Ca(2+) binding in members of the superfamily, particularly in vertebrates, is due to the selective presence of unfavorable amino acids (largely Arg) at key Ca(2+)-ligation positions and that engineering of the canonical Ca(2+)-binding residues can confer binding activity on an otherwise inactive domain. Through this work, we demonstrate that betagamma-crystallins with the N/D-N/D-#-I-S/T-S motif form an extensive set of Ca(2+)-binding proteins prevalent in all of the three kingdoms of life.

Calcium-binding to lens βB2- and βA3-crystallins suggests that all β-crystallins are calcium-binding proteins

Crystallins are the major proteins of a mammalian eye lens. The topologically similar eye lens proteins, β‐ and γ‐crystallins, are the prototype and founding members of the βγ‐crystallin superfamily. βγ‐Crystallins have until recently been regarded as structural proteins. However, the calcium‐binding properties of a few members and the potential role of βγ‐crystallins in fertility are being investigated. Because the calcium‐binding elements of other member proteins, such as spherulin 3a, are not present in βB2‐crystallin and other βγ‐crystallins from fish and mammalian genomes, it was argued that lens βγ‐crystallins should not bind calcium. In order to probe whether β‐crystallins can bind calcium, we selected one basic (βB2) and one acidic (βA3) β‐crystallin for calcium‐binding studies. Using calcium‐binding assays such as 45Ca overlay, terbium binding, Stains‐All and isothermal titration calorimetry, we established that both βB2‐ and βA3‐crystallin bind calcium with moderate affinity. There was no significant change in their conformation upon binding calcium as monitored by fluorescence and circular dichroism spectroscopy. However, 15N‐1H heteronuclear single quantum correlation NMR spectroscopy revealed that amide environment of several residues underwent changes indicating calcium ligation. With the corroboration of calcium‐binding to βB2‐ and βA3‐crystallins, we suggest that all β‐crystallins bind calcium. Our results have important implications for understanding the calcium‐related cataractogenesis and maintenance of ionic homeostasis in the lens.

Solution Structure and Calcium-Binding Properties of M-Crystallin, A Primordial βγ-Crystallin from Archaea

The lens betagamma-crystallin superfamily has many diverse but topologically related members belonging to various taxa. Based on structural topology, these proteins are considered to be evolutionarily related to lens crystallins, suggesting their origin from a common ancestor. Proteins with betagamma-crystallin domains, although found in some eukaryotes and eubacteria, have not yet been reported in archaea. Sequence searches in the genome of the archaebacterium Methanosarcina acetivorans revealed the presence of a protein annotated as a betagamma-crystallin family protein, named M-crystallin. Solution structure of this protein indicates a typical betagamma-crystallin fold with a paired Greek-key motif. Among the known structures of betagamma-crystallin members, M-crystallin was found to be structurally similar to the vertebrate lens betagamma-crystallins. The Ca(2+)-binding properties of this primordial protein are somewhat more similar to those of vertebrate betagamma-crystallins than to those of bacterial homologues. These observations, taken together, suggest that amphibian and vertebrate betagamma-crystallin domains are evolutionarily more related to archaeal homologues than to bacterial homologues. Additionally, identification of a betagamma-crystallin homologue in archaea allows us to demonstrate the presence of this domain in all the three domains of life.

Calcium-binding Crystallins from Yersinia pestis CHARACTERIZATION OF TWO SINGLE βγ-CRYSTALLIN DOMAINS OF A PUTATIVE EXPORTED PROTEIN

βγ-Crystallin is a superfamily with diverse members from vertebrate lens to microbes. However, not many members have been identified and studied. Here, we report the identification of a putative exported protein from Yersinia pestis as a member of the βγ-crystallin superfamily. Even though calcium has been known to play an important role in the physiology and virulence of the Yersinia genus, calcium-binding proteins have not yet been identified. We have studied the calcium-binding properties of two of the three crystallin domains present in this putative exported protein designated “Yersinia crystallin.” These two domains (D1 and D2) have unique AA and BB types of arrangement of their Greek key motifs unlike the domains of other members of the βγ-crystallin superfamily, which are either AB or BA types. These domains bind two calcium ions with low and high affinity-binding sites. We showed their calcium-binding properties using various probes for calcium and the effect of calcium on their secondary and tertiary structures. Although both domains bind calcium, D1 underwent drastic changes in secondary and tertiary structure and hydrodynamic volume upon calcium binding. Domain D1, which is intrinsically unstructured in the apo form, requires calcium for the typical βγ-crystallin fold. Calcium exerted an extrinsic stabilization effect on domain D1 but not on D2, which is also largely unstructured. We suggest that this protein might be involved in calcium-dependent processes, such as stress response or physiology in the Yersinia genus, similar to its microbial relatives and mammalian lens crystallins.

Equilibrium Unfolding of Neuronal Calcium Sensor-1 N-TERMINAL MYRISTOYLATION INFLUENCES UNFOLDING AND REDUCES PROTEIN STIFFENING IN THE PRESENCE OF CALCIUM

Neuronal calcium sensor-1 (NCS-1), a Ca2+-binding protein of the calcium sensor family, modulates various functions in intracellular signaling pathways. The N-terminal glycine in this protein is myristoylated, which is presumably necessary for its physiological functions. In order to understand the structural role of myristoylation and calcium on conformational stability, we have investigated the equilibrium unfolding and refolding of myristoylated and non-myristoylated NCS-1. The unfolding of these two forms of NCS-1 in the presence of calcium is best characterized by a five-state equilibrium model, and multiple intermediates accumulate during unfolding. Calcium exerts an extrinsic stabilizing effect on both forms of the protein. In the absence of calcium, the stability of both forms is dramatically decreased, and the unfolding follows a four-state equilibrium model. The equilibrium transitions are fully reversible in the presence of calcium. Myristoylation affects the pattern of equilibrium transitions substantially but not the number of intermediates, suggesting a structural role. Our data suggest that myristoylation reduces the stiffening of the protein during initial unfolding in the presence of calcium. The effects of myristoylation are more pronounced when calcium is present, suggesting a relationship between them. Inactivating the third EF-hand motif (E120Q mutant) drastically affects the equilibrium unfolding transitions, and calcium has no effect on these transitions of the mutants. The unfolding transitions of both forms of the mutant are similar to the transitions followed by the apo forms of myristoylated and non-myristoylated NCS-1. These results suggest that the role of myristoylation in unfolding/refolding of the protein is largely dependent on the presence of calcium.

Calcium and chlorpromazine binding to the EF-hand peptides of neuronal calcium sensor-1

Neuronal calcium sensor-1, a protein of calcium sensor family, is known to have four structural EF-hands. We have synthesised peptides corresponding to all the four EF-hands and studied their conformation and calcium-binding. Our data confirm that the first putative site, a non-canonical one (EF1), does not bind calcium. We have investigated if this lack of binding is due to the presence of non-favoured residues (particularly at +x and -z co-ordinating positions) of the loop. We have mutated these residues and found that after modification the peptides bound calcium. However, these mutated peptides (EF1 and its functional mutants) do not show any Ca(2+) induced changes in far-UV CD. EF2, EF3, and EF4 peptides bind Ca(2+), EF3 being the strongest binder, followed by EF4. Our data of Ca(2+)-binding to individual EF peptides show that there are three active Ca(2+)-binding sites in NCS-1. We have also studied the binding of a neuroleptic drug, chlorpromazine, with the protein as well as with its EF-hands. CPZ binds myristoylated as well as non-myristoylated NCS-1 in Ca(2+)-dependent manner, with dynamic interaction to myristoylated protein. CPZ does not bind to EF1, but binds to functional EF-hand peptides and induces changes in far-UV CD. Our results suggest that NCS-1 could be a target of such antipsychotic and neuroleptic drugs.

Rapid purification of recombinant βB2-crystallin using hydrophobic interaction chromatography

BetaB2-crystallin, the major subunit of beta-crystallins, is difficult to purify either from lens homogenate or from betaH-or betaL-crystallins. It has been prepared by heterologous expression in Escherichia coli. Most often, the methods used for purifying a recombinant globular protein employ the combination of ion-exchange with gel filtration chromatography. In the case of betaB2-crystallin too, different approaches have been used to obtain the purified protein, majority of which use a combination of ion-exchange and gel filtration chromatography. We present a new approach to purify betaB2-crystallin using hydrophobic interaction chromatography. In this method, the protein is bound to the hydrophobic matrix in the presence of high concentration of a non-chaotropic salt and eluted by decreasing the salt concentration. The method that we have used for the purification of this globular protein has definite advantages over the earlier methods in its simplicity and efficiency. The most noted advantage of this procedure is the rapid purification with a relatively purified product and a comparatively high yield (>20 mg/L of culture). Over all, the present protocol provides a rapid, efficient and simplified procedure for the preparation of betaB2-crystallin in large yield, sufficient for structural and functional studies.