The N-terminal domain of betaB2-crystallin resembles the putative ancestral homodimer

betagamma-crystallins from the eye lens are proteins consisting of two similar domains joined by a short linker. All three-dimensional structures of native proteins solved so far reveal similar pseudo-2-fold pairing of the domains reflecting their presumed ancient origin from a single-domain homodimer. However, studies of engineered single domains of members of the betagamma-crystallin superfamily have not revealed a prototype ancestral solution homodimer. Here we report the 2.35 A X-ray structure of the homodimer of the N-terminal domain of rat betaB2-crystallin (betaB2-N). The two identical domains pair in a symmetrical manner very similar to that observed in native betagamma-crystallins, where N and C-terminal domains (which share approximately 35% sequence identity) are related by a pseudo-2-fold axis. betaB2-N thus resembles the ancestral prototype of the betagamma-crystallin superfamily as it self-associates in solution to form a dimer with an essentially identical domain interface as that between the N and C domains in betagamma-crystallins, but without the benefit of a covalent linker. The structure provides further evidence for the role of two-domain pairing in stabilising the protomer fold. These results support the view that the betagamma-crystallin superfamily has evolved by a series of gene duplication and fusion events from a single-domain ancestor capable of forming homodimers.     

Stability,homodimerization, and calcium-binding properties of a single,variant betagamma-crystallin domain of the protein absent in melanoma 1 (AIM1)

AIM1 (absent in melanoma), a candidate suppressor of malignancy in melanoma, is a nonlens member of the betagamma-crystallin superfamily, which contains six predicted betagamma domains. The first betagamma-crystallin domain of AIM1 (AIM1-g1) diverges most in sequence from the superfamily consensus. To examine its ability to fold and behave like a normal betagamma domain, we cloned AIM1-g1 and overexpressed it in Escherichia coli as a recombinant protein. The recombinant domain was found to be a stable, soluble protein, similar to lens protein gammaBeta-crystallin in secondary structure. The tertiary structure of AIM1-g1 is dominated by the contribution of aromatic amino acids and cysteine. AIM1-g1 undergoes concentration-independent, noncovalent homodimerization with no trace of monomer, similar to a one-domain protein spherulin 3a. Since many betagamma domain proteins bind calcium, we have also investigated the calcium-binding properties of AIM1-g1 by various methods. AIM1-g1 binds the calcium-mimic dye Stains-all, the calcium probe terbium (with K(D) 170 microM), and (45)Ca when blotted on a membrane. AIM1-g1 binds calcium (K(D) 30 microM) with a comparatively higher affinity than bovine lens gamma-crystallin (90 microM). However, calcium binding does not induce significant change in the protein conformation in the near- and far-UV CD and in fluorescence. The AIM1-g1 domain is as stable as domains of betagamma-crystallins (betaB2- or gammaS-crystallins) as monitored by guanidinium chloride unfolding (midpoint of unfolding transition is 1.8 M GdmCl), and the stability of the protein is not altered upon binding calcium as evaluated by equilibrium unfolding. These results show that, despite the sequence variation, AIM1-g1 folds such as a betagamma domain, binds calcium and undergoes dimerization.     

Three-dimensional domain swapping in nitrollin, a single-domain betagamma-crystallin from Nitrosospira multiformis, controls protein conformation and stability but not dimerization

The betagamma-crystallin superfamily has a well-characterized protein fold, with several members found in both prokaryotic and eukaryotic worlds. A majority of them contain two betagamma-crystallin domains. A few examples, such as ciona crystallin and spherulin 3a exist that represent the eukaryotic single-domain proteins of this superfamily. This study reports the high-resolution crystal structure of a single-domain betagamma-crystallin protein, nitrollin, from the ammonium-oxidizing soil bacterium Nitrosospira multiformis. The structure retains the characteristic betagamma-crystallin fold despite a very low sequence identity. The protein exhibits a unique case of homodimerization in betagamma-crystallins by employing its N-terminal extension to undergo three-dimensional (3D) domain swapping with its partner. Removal of the swapped strand results in partial loss of structure and stability but not dimerization per se as determined using gel filtration and equilibrium unfolding studies. Overall, nitrollin represents a distinct single-domain prokaryotic member that has evolved a specialized mode of dimerization hitherto unknown in the realm of betagamma-crystallins.     

Circular permutation of betaB2-crystallin changes the hierarchy of domain assembly.

The betagamma-crystallins form a superfamily of eye lens proteins comprised of multiple Greek motifs that are symmetrically organized into domains and higher assemblies. In the betaB2-crystallin dimer each polypeptide folds into two similar domains that are related to monomeric gamma-crystallin by domain swapping. The crystal structure of the circularly permuted two-domain betaB2 polypeptide shows that permutation converts intermolecular domain pairing into intramolecular pairing. However, the dimeric permuted protein is, in fact, half a native tetramer. This result shows how the sequential order of domains in multi-domain proteins can affect quaternary domain assembly.     

Kinetic and thermodynamic stabilization of the betagamma-crystallin homolog spherulin 3a from Physarum polycephalum by calcium binding.

Globular proteins may be stabilized, either intrinsically, at the various levels of the structural hierarchy, or extrinsically, by ligand binding. In the case of the dormant all-beta protein spherulin 3a (S3a) from the slime mold Physarum polycephalum, binding of calcium ions causes extreme kinetic and thermodynamic stabilization. S3a is the only known single-domain member of the two Greek key superfamily of betagamma-crystallins sharing the extreme long-term stability of its homologs in vertebrate eye lens. Spectral analysis allows two Ca2+-binding sites with KD=9 microM and 200 microM to be distinguished. Unfolding in the absence and in the presence of Ca2+gives evidence for extreme kinetic stabilization of the protein: In the absence of Ca2+, the half-time of unfolding in 2. 5 M guanidinium chloride (GdmCl) equals 8.3 minutes, whereas in the presence of Ca2+, even in 7.5 M GdmCl, it exceeds nine hours. To reach the equilibrium of unfolding in the absence and in the presence of Ca2+takes one day and eight weeks, respectively. The corresponding Gibbs free energies (based on the two-state model) are 77 and 135 kJ/mol. Saturation of S3a with Ca2+leads to an upward shift of the temperature-induced equilibrium transition by ca 20 deg. C. The in situ Ca2+concentration in the spherules is sufficient for the complete complexation of S3a in vivo.     

Urochordate betagamma-crystallin and the evolutionary origin of the vertebrate eye lens

A refracting lens is a key component of our image-forming camera eye; however, its evolutionary origin is unknown because precursor structures appear absent in nonvertebrates. The vertebrate betagamma-crystallin genes encode abundant structural proteins critical for the function of the lens. We show that the urochordate Ciona intestinalis, which split from the vertebrate lineage before the evolution of the lens, has a single gene coding for a single domain monomeric betagamma-crystallin. The crystal structure of Ciona betagamma-crystallin is very similar to that of a vertebrate betagamma-crystallin domain, except for paired, occupied calcium binding sites. The Ciona betagamma-crystallin is only expressed in the palps and in the otolith, the pigmented sister cell of the light-sensing ocellus. The Ciona betagamma-crystallin promoter region targeted expression to the visual system, including lens, in transgenic Xenopus tadpoles. We conclude that the vertebrate betagamma-crystallins evolved from a single domain protein already expressed in the neuroectoderm of the prevertebrate ancestor. The conservation of the regulatory hierarchy controlling betagamma-crystallin expression between organisms with and without a lens shows that the evolutionary origin of the lens was based on co-option of pre-existing regulatory circuits controlling the expression of a key structural gene in a primitive light-sensing system.     

The X-ray crystal structure of human gamma S-crystallin C-terminal domain.

gammaS-crystallin is a major human lens protein found in the outer region of the eye lens, where the refractive index is low. Because crystallins are not renewed they acquire post-translational modifications that may perturb stability and solubility. In common with other members of the betagamma-crystallin superfamily, gammaS-crystallin comprises two similar beta-sheet domains. The crystal structure of the C-terminal domain of human gammaS-crystallin has been solved at 2.4 A resolution. The structure shows that in the in vitro expressed protein, the buried cysteines remain reduced. The backbone conformation of the "tyrosine corner" differs from that of other betagamma-crystallins because of deviation from the consensus sequence. The two C-terminal domains in the asymmetric unit are organized about a slightly distorted 2-fold axis to form a dimer with similar geometry to full-length two-domain family members. Two glutamines found in lattice contacts may be important for short range interactions in the lens. An asparagine known to be deamidated in human cataract is located in a highly ordered structural region.     

Superfamilies SDR and MDR: From early ancestry to present forms. Emergence of three lines, a Zn-metalloenzyme, and distinct variabilities

Two large gene and protein superfamilies, SDR and MDR (short- and medium-chain dehydrogenases/reductases), were originally defined from analysis of alcohol and polyol dehydrogenases. The superfamilies contain minimally 82 and 25 genes, respectively, in humans, minimally 324 and 86 enzyme families when known lines in other organisms are also included, and over 47,000 and 15,000 variants in existing sequence data bank entries. SDR enzymes have one-domain subunits without metal and MDR two-domain subunits without or with zinc, and these three lines appear to have emerged in that order from the universal cellular ancestor. This is compatible with their molecular architectures, present multiplicity, and overall distribution in the kingdoms of life, with SDR also of viral occurrence. An MDR-zinc, when present, is often, but not always, catalytic. It appears also to have a structural role in inter-domain interactions, coenzyme binding and substrate pocket formation, as supported by domain variability ratios and ligand positions. Differences among structural and catalytic zinc ions may be relative and involve several states. Combined, the comparisons trace evolutionary properties of huge superfamilies, with partially redundant enzymes in cellular redox functions.     

2.4 A resolution crystal structure of the prototypical hormone-processing protease Kex2 in complex with an Ala-Lys-Arg boronic acid inhibitor

This paper reports the first structure of a member of the Kex2/furin family of eukaryotic pro-protein processing proteases, which cleave sites consisting of pairs or clusters of basic residues. Reported is the 2.4 A resolution crystal structure of the two-domain protein ssKex2 in complex with an Ac-Ala-Lys-boroArg inhibitor (R = 20.9%, R(free) = 24.5%). The Kex2 proteolytic domain is similar in its global fold to the subtilisin-like superfamily of degradative proteases. Analysis of the complex provides a structural basis for the extreme selectivity of this enzyme family that has evolved from a nonspecific subtilisin-like ancestor. The P-domain of ssKex2 has a novel jelly roll like fold consisting of nine beta strands and may potentially be involved, along with the buried Ca(2+) ion, in creating the highly determined binding site for P(1) arginine.     

Evolution of a protein superfamily: Relationships between vertebrate lens crystallins and microorganism dormancy proteins

Summary A search of sequence databases shows that spherulin 3a, an encystment-specific protein ofPhysarum polycephalum, is probably structurally related to the - and -crystallins, vertebrate ocular lens proteins, and to Protein S, a sporulation-specific protein ofMyxococcus xanthus. The - and -crystallins have two similar domains thought to have arisen by two successive gene duplication and fusion events. Molecular modeling confirms that spherulin 3a has all the characteristics required to adopt the tertiary structure of a single -crystallin domain. The structure of spherulin 3a thus illustrates an earlier stage in the evolution of this protein superfamily. The relationship of - and -crystallins to spherulin 3a and Protein S suggests that the lens proteins were derived from an ancestor with a role in stressresponse, perhaps a response to osmotic stress.     

Structural comparison of cupredoxin domains: domain recycling to construct proteins with novel functions.

The three-dimensional structures of the copper-containing enzymes ascorbate oxidase, ceruloplasmin, and nitrite reductase, comprised of multiple domains with a cupredoxin fold, are consistent with having evolved from a common ancestor. The presence or absence of copper sites has complicated ascertaining the structural and evolutionary relationship among these and related proteins. Simultaneous structural superposition of the enzyme domains and their known cupredoxin relatives shows clearly that there are at least six cupredoxin classes, and that the evolution of the conserved core of these domains is independent of the presence or absence of copper sites. Relationships among the variable loops in these structures show that the two-domain ancestor of the blue oxidases contained a trinuclear-copper interface but could not have functioned in a monomeric state. Comparison of the sequence of the copper-containing, iron-regulating protein. Ferrous transport (Fet3) from yeast to the structurally defined core and loop residues of the cupredoxins suggests specific residues that could be involved in the ferroxidase activity of Fet3.     

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

The lens βγ-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 βγ-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 βγ-crystallin family protein, named M-crystallin. Solution structure of this protein indicates a typical βγ-crystallin fold with a paired Greek-key motif. Among the known structures of βγ-crystallin members, M-crystallin was found to be structurally similar to the vertebrate lens βγ-crystallins. The Ca^2 +-binding properties of this primordial protein are somewhat more similar to those of vertebrate βγ-crystallins than to those of bacterial homologues. These observations, taken together, suggest that amphibian and vertebrate βγ-crystallin domains are evolutionarily more related to archaeal homologues than to bacterial homologues. Additionally, identification of a βγ-crystallin homologue in archaea allows us to demonstrate the presence of this domain in all the three domains of life.     

Reassembly of wall domains of Roman-snail (Helix pomatia) beta-haemocyanin.

beta-Haemocyanin molecules consist of 20 very large polypeptide chains. These chains are composed of eight structural domains. So-called 'collar' domains can be removed by trypsinolysis of the native cylindrical molecule, resulting in an association of the remaining hollow cylinders into large tubular polymers. Dissociation of the tubular polymers gives one single- and four multi-domain fragments. The role of these fragments in the reassembly process of these tubular polymers was investigated. The two-domain fragment could form tubular polymers. The other domain fragments were not able to form tubular polymers unless in the presence of the two-domain fragment. Tubular polymers with enlarged diameter and ribbon-like structures were observed in the reassembly products when the one-domain fragment was omitted.     

Calcium-binding to lens betaB2- and betaA3-crystallins suggests that all beta-crystallins are calcium-binding proteins

Crystallins are the major proteins of a mammalian eye lens. The topologically similar eye lens proteins, beta- and gamma-crystallins, are the prototype and founding members of the betagamma-crystallin superfamily. Betagamma-crystallins have until recently been regarded as structural proteins. However, the calcium-binding properties of a few members and the potential role of betagamma-crystallins in fertility are being investigated. Because the calcium-binding elements of other member proteins, such as spherulin 3a, are not present in betaB2-crystallin and other betagamma-crystallins from fish and mammalian genomes, it was argued that lens betagamma-crystallins should not bind calcium. In order to probe whether beta-crystallins can bind calcium, we selected one basic (betaB2) and one acidic (betaA3) beta-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 betaB2- and betaA3-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 betaB2- and betaA3-crystallins, we suggest that all beta-crystallins bind calcium. Our results have important implications for understanding the calcium-related cataractogenesis and maintenance of ionic homeostasis in the lens.     

Unfolding crystallins: the destabilizing role of a beta-hairpin cysteine in betaB2-crystallin by simulation and experiment

The thermodynamic and kinetic stabilities of the eye lens family of betagamma-crystallins are important factors in the etiology of senile cataract. They control the chance of proteins unfolding, which can lead to aggregation and loss of transparency. betaB2-Crystallin orthologs are of low stability and comprise two typical betagamma-crystallin domains, although, uniquely, the N-terminal domain has a cysteine in one of the conserved folded beta-hairpins. Using high-temperature (500 K) molecular dynamics simulations with explicit solvent on the N-terminal domain of rodent betaB2-crystallin, we have identified in silico local flexibility in this folded beta-hairpin. We have shown in vitro using two-domain human betaB2-crystallin that replacement of this cysteine with a more usual aromatic residue (phenylalanine) results in a gain in conformational stability and a reduction in the rate of unfolding. We have used principal components analysis to visualize and cluster the coordinates from eight separate simulated unfolding trajectories of both the wild-type and the C50F mutant N-terminal domains. These data, representing fluctuations around the native well, show that although the mutant and wild-type appear to behave similarly over the early time period, the wild type appears to explore a different region of conformational space. It is proposed that the advantage of having this low-stability cysteine may be correlated with a subunit-exchange mechanism that allows betaB2-crystallin to interact with a range of other beta-crystallin subunits.     

Solution structure of the flexible class II ubiquitin-conjugating enzyme Ubc1 provides insights for polyubiquitin chain assembly

E2 conjugating enzymes form a thiol ester intermediate with ubiquitin, which is subsequently transferred to a substrate protein targeted for degradation. While all E2 proteins comprise a catalytic domain where the thiol ester is formed, several E2s (class II) have C-terminal extensions proposed to control substrate recognition, dimerization, or polyubiquitin chain formation. Here we present the novel solution structure of the class II E2 conjugating enzyme Ubc1 from Saccharomyces cerevisiae. The structure shows the N-terminal catalytic domain adopts an alpha/beta fold typical of other E2 proteins. This domain is physically separated from its C-terminal domain by a 22-residue flexible tether. The C-terminal domain adopts a three-helix bundle that we have identified as an ubiquitin-associated domain (UBA). NMR chemical shift perturbation experiments show this UBA domain interacts in a regioselective manner with ubiquitin. This two-domain structure of Ubc1 was used to identify other UBA-containing class II E2 proteins, including human E2-25K, that likely have a similar architecture and to determine the role of the UBA domain in facilitating polyubiquitin chain formation.     

Three-Dimensional Domain Swapping in Nitrollin, a Single-Domain βγ-Crystallin from Nitrosospira multiformis, Controls Protein Conformation and Stability but Not Dimerization

The βγ-crystallin superfamily has a well-characterized protein fold, with several members found in both prokaryotic and eukaryotic worlds. A majority of them contain two βγ-crystallin domains. A few examples, such as ciona crystallin and spherulin 3a exist that represent the eukaryotic single-domain proteins of this superfamily. This study reports the high-resolution crystal structure of a single-domain βγ-crystallin protein, nitrollin, from the ammonium-oxidizing soil bacterium Nitrosospira multiformis. The structure retains the characteristic βγ-crystallin fold despite a very low sequence identity. The protein exhibits a unique case of homodimerization in βγ-crystallins by employing its N-terminal extension to undergo three-dimensional (3D) domain swapping with its partner. Removal of the swapped strand results in partial loss of structure and stability but not dimerization per se as determined using gel filtration and equilibrium unfolding studies. Overall, nitrollin represents a distinct single-domain prokaryotic member that has evolved a specialized mode of dimerization hitherto unknown in the realm of βγ-crystallins.     

Kinetic properties and structural characteristics of an unusual two-domain arginine kinase of the clam Corbicula japonica

Arginine kinase (AK) from the clam Corbicula japonica is a unique enzyme in that it has an unusual two-domain structure with molecular mass of 80 kDa. It lacks two functionally important amino acid residues, Asp-62 and Arg-193, which are conserved in other 40 kDa AKs and are assumed to be key residues for stabilizing the substrate-bound structure. K m arg and Vmax values for the recombinant two-domain AK were determined. These values were close to those of usual 40 kDa AKs, although Corbicula AK lacks the functionally important Asp-62 and Arg-193. Domain 2 of Corbicula AK was separated from the two-domain enzyme and was expressed in Escherichia coli. Domain 2 still exhibited activity. However, kinetic parameters for domain 2 appeared to be slightly, but significantly, different from those of two-domain AK. Thus, it is likely that the formation of the contiguous dimer alters the kinetic properties of its constituent domains significantly. Comparison of K d arg and K m arg for two-domain AK and its domain 2 showed that the affinity of the enzyme for arginine is greater in the presence of substrate ATP than in its absence. Presumably this difference is correlated with the large structural differences in the enzyme in the presence or absence of substrate, namely open and closed structures. We expressed three mutants of Corbicula AK domain 2 (His-60 to Gly or Arg, Asp-197 to Gly), and determined their K m arg and Vmax values. The affinity for the substrate arginine in mutant enzymes was reduced considerably, accompanied by a decrease in Vmax. These results suggest that His-60 and Asp-197 affect the substrate binding system, and are consistent with the hypothesis that a hydrogen bond is formed between His-60 and Asp-197 in Corbicula AK as a substitute for the Asp-62 and Arg-193 bond in normal AKs.