Aggregation-prone near-native intermediate formation during unfolding of a structurally similar nonlenticular ßγ-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 ßγ-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 ßγ-crystallin domain of non-lens vertebrate protein Crybg3 from mouse brain. The crystal structure determined at 1.86 Å demonstrates that the ßγ-crystallin domain of Crybg3 resembles more closely the lens ßγ-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 ßγ-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.

The betagamma-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.

Crystal structure of a fungal protease inhibitor from Antheraea mylitta.

Indian tasar silk is produced by a wild insect called Antheraea mylitta. Insects do not have any antigen-antibody mediated immune system like vertebrates but they produce a wide variety of effector proteins and peptides possessing potent antifungal and antibacterial activity to combat microbial attack. Antheraea mylitta expresses a fungal protease inhibitor AmFPI-1, in the hemolymph that inhibits alkaline protease of Aspergillus oryzae for protection against fungal infection. AmFPI-1 is purified from the hemolymph, crystallized and the structure is solved using the single isomorphous replacement with anomalous scattering (SIRAS) method to a resolution of 2.1 A. AmFPI-1 is a single domain protein possessing a unique fold that consists of three helices and five beta strands stabilized by a network of six disulfide bonds. The reactive site of AmFPI-1 is located in the loop formed by residues 46-66, wherein Lys54 is the P(1) residue. Superimposition of the loop with reactive sites of other canonical protease inhibitors shows that reactive site conformation of AmFPI-1 is similar to them. The structure of AmFPI-1 provides a framework for the docking of a 1:1 complex between AmFPI-1 and alkaline protease. This study addresses the structural basis of AmFPI-1's specificity towards a fungal serine protease but not to mammalian trypsin and may help in designing specific inhibitors against fungal proteases.

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.

Exploring the limits of sequence and structure in a variant betagamma-crystallin domain of the protein absent in melanoma-1 (AIM1).

Betagamma-crystallins belong to a superfamily of proteins in prokaryotes and eukaryotes that are based on duplications of a characteristic, highly conserved Greek key motif. Most members of the superfamily in vertebrates are structural proteins of the eye lens that contain four motifs arranged as two structural domains. Absent in melanoma 1 (AIM1), an unusual member of the superfamily whose expression is associated with suppression of malignancy in melanoma, contains 12 betagamma-crystallin motifs in six domains. Some of these motifs diverge considerably from the canonical motif sequence. AIM1g1, the first betagamma-crystallin domain of AIM1, is the most variant of betagamma-crystallin domains currently known. In order to understand the limits of sequence variation on the structure, we report the crystal structure of AIM1g1 at 1.9 A resolution. Despite having changes in key residues, the domain retains the overall betagamma-crystallin fold. The domain also contains an unusual extended surface loop that significantly alters the shape of the domain and its charge profile. This structure illustrates the resilience of the betagamma fold to considerable sequence changes and its remarkable ability to adapt for novel functions.

Regulatory and structural EF-hand motifs of neuronal calcium sensor-1: Mg 2+ modulates Ca 2+ binding, Ca 2+ -induced conformational changes, and equilibrium unfolding transitions.

Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca(2+) signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca(2+)-specific) and structural (Ca(2+)- or Mg(2+)-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca(2+) binding using NMR and EF-hand mutants. Ca(2+) titration, as monitored by [(15)N,(1)H] heteronuclear single quantum coherence, suggests that Ca(2+) binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg(2+) binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca(2+)-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca(2+) and not Mg(2+). Ca(2+) binding induces conformational rearrangements in the protein by reversing Mg(2+)-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg(2+) with Ca(2+) reduces the Ca(2+)-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca(2+)-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg(2+)-bound NCS-1 are similar, the early unfolding transitions of Ca(2+)-bound NCS-1 are partially influenced in the presence of Mg(2+). This study demonstrates the importance of Mg(2+) as a modulator of calcium homeostasis and active-state behavior of NCS-1.

Neuronal Ca2+ signaling via caldendrin and calneurons.

The calcium sensor protein caldendrin is abundantly expressed in neurons and is thought to play an important role in different aspects of synapto-dendritic Ca2+ signaling. Caldendrin is highly abundant in the postsynaptic density of a subset of excitatory synapses in brain and its distinct localization raises several decisive questions about its function. Previous work suggests that caldendrin is tightly associated with Ca2+ - and Ca2+ release channels and might be involved in different aspects of the organization of the postsynaptic scaffold as well as with synapse-to-nucleus communication. In this report we introduce two new EF-hand calcium sensor proteins termed calneurons that apart from calmodulin represent the closest homologues of caldendrin in brain. Calneurons have a different EF-hand organization than other calcium sensor proteins, are prominently expressed in neurons and will presumably bind Ca2+ with higher affinity than caldendrin. Despite some significant structural differences it is conceivable that they are involved in similar Ca2+ regulated processes like caldendrin and neuronal calcium sensor proteins.

Crystallization and preliminary X-ray diffraction analysis of a protease inhibitor from the haemolymph of the Indian tasar silkworm Antheraea mylitta.

A protein with inhibitory activity against fungal proteases was purified from the haemolymph of the Indian tasar silkworm Antheraea mylitta and was crystallized using the hanging-drop vapour-diffusion method. Polyethylene glycol 3350 was used as a precipitant. Crystals belonged to space group P6(3)22, with unit-cell parameters a = b = 60.6, c = 85.1 angstroms. X-ray diffraction data were collected and processed to a maximum resolution of 2.1 angstroms.

Crystallization and preliminary X-ray crystallographic investigations on a betagamma-crystallin domain of absent in melanoma 1 (AIM1), a protein from Homo sapiens.

AIM1g1 is a single betagamma-crystallin domain from the protein absent in melanoma 1 (AIM1), which appears to play a role in the suppression of melanomas. This domain is known to bind calcium and its structure would help in identifying calcium-coordinating sites in vertebrate crystallins, which have hitherto been believed to have lost this ability during evolution. Crystallization of this domain was performed by the hanging-drop vapour-diffusion method. Crystals diffracted to a maximum resolution of 1.86 A and were found to belong to space group P6(1) or P6(5), with unit-cell parameters a = b = 54.98, c = 59.73 A. Solvent-content analysis indicated the presence of one monomer per asymmetric unit.