Hypomagnesemia with Secondary Hypoparathyroidism and Hypocalcemia due to Novel Variants in the Transient Receptor Potential Cation Channel Subfamily M Member 6 ( TRPM6 ) Gene.

HOMG1 (hypomagnesemia 1, intestinal) or hypomagnesemia with secondary hypocalcemia is a rare autosomal recessive disorder of magnesium metabolism, characterized by impaired magnesium absorption. This disorder may mimic other conditions presenting with neonatal seizures. Here, we report an infant diagnosed to have hypomagnesemia with secondary hypocalcemia due to novel variants in TRPM6 gene.

Structural and functional characterization of a putative carbonic anhydrase from Geobacillus kaustophilus reveals its cambialistic function.

Carbonic anhydrases (CA) are the most ubiquitous ancient zinc metalloenzymes known. Here we report the structural and functional analysis of a hypothetical protein GK2848 from Geobacillus kaustophilus. The analysis revealed that it belongs to the γ-class of CA (termed as Cag). Only a limited number of γ-class CA's have been characterized till date. Interestingly Cag contains magnesium at its active site instead of a traditional zinc ion. Based on the structural and sequence comparison with similar γ-CA's the putative active site residues of Cag were identified. This analysis revealed that an important catalytic residue and a proton shuttle residue (Glu62 and Glu84 respectively) of Cam (previously characterized γ-CA from Methanosarcina thermophila) are absent in Cag, however certain other active site residues are conserved both in Cag and Cam. This suggests that Cag uses a different set of residues for the reversible hydration of CO2 to HCO3- when compared with Cam. Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES) and 25Mg and 67Zn NMR studies on Cag and its mutants revealed that either Mg or Zn can occupy the active site which suggests the cambialistic nature of the enzyme.

Molecular dynamics simulation of metal free structure of Lmb, a laminin-binding adhesin of Streptococcus agalactiae: metal removal and its structural implications.

Metal-binding receptors are one of the extracellular components of ATP-binding cassette transporters that are essential for regulation of metal homeostasis in bacteria. Laminin-binding adhesin (Lmb) of Streptococcus agalactiae falls under this class of solute binding proteins. It binds to zinc with a high affinity. Crystal structure of Lmb solved previously by our group reveals that the zinc is tetrahedrally coordinated by three histidines and a glutamate at the interdomain cleft. Lmb contains a long disordered loop close to the metal-binding site whose precise function is unknown. Several experimental attempts to produce apo-Lmb failed and this prompted us to carry out in silico studies to analyse the structural importance of the metal in Lmb. Here, we present the results of the molecular dynamics (MD) simulation studies of native, apo-(metal removed) and the long loop truncated Lmb models along with a homologous protein, TroA from Treponema pallidum that was taken up for validating the MD results of Lmb. Absence of a metal results in significant structural changes in Lmb, particularly at the metal-binding pocket and with the long loop, although the overall fold is retained. This study thus revealed that the Lmb can exist in different conformational states with subtle differences in the overall fold based on the presence or absence of the metal. This could be functionally important for a putative metal uptake and release and also for the adhesive function of Lmb in recognizing laminin, which contains a high number of zinc finger motifs.

Crystal structure of Aquifex aeolicus gene product Aq1627: a putative phosphoglucosamine mutase reveals a unique C-terminal end-to-end disulfide linkage.

The Aq1627 gene from Aquifex aeolicus, a hyperthermophilic bacterium has been cloned and overexpressed in Escherichia coli. The protein was purified to homogeneity and its X-ray crystal structure was determined to 1.3 Å resolution using multiple wavelength anomalous dispersion phasing. The structural and sequence analysis of Aq1627 is suggestive of a putative phosphoglucosamine mutase. The structural features of Aq1627 further indicate that it could belong to a new subclass of the phosphoglucosamine mutase family. Aq1627 structure contains a unique C-terminal end-to-end disulfide bond, which links two monomers and this structural information can be used in protein engineering to make proteins more stable in different applications.

Isopeptide bond in collagen- and fibrinogen-binding MSCRAMMs.

The internal isopeptide bonds are amide bonds formed autocatalytically between the side chains of Lys and Asn/Asp residues and have been discovered recently. These bonds are well conserved in Gram-positive bacterial pilin proteins and are also observed over a wide range of Gram-positive bacterial surface proteins. The presence of these bonds confers the pilus subunits with remarkable properties in terms of thermal stability and resistance to proteases. Like pili, microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) are also surface proteins found only in Gram-positive bacteria. They specifically interact with the extracellular matrix (ECM) molecules like collagen, fibrinogen, fibronectin, laminin, etc. Many biophysical and biochemical studies have been carried out to characterize the isopeptide bonds in pili proteins from Gram-positive bacteria, but no attempts have been made to study the isopeptide bonds in MSCRAMMs. This short review aims to study the significance of the isopeptide bonds in relation to their function, by analyzing the crystal structures of collagen- and fibrinogen-binding MSCRAMMs. In this analysis, interestingly, we observed that the putative isopeptide bonds are restricted to the collagen-binding MSCRAMMs. Based on analogy with bacterial pilus subunits, we hypothesize that the collagen-binding MSCRAMMs possessing putative isopeptide bonds exhibit similar structural properties, which could help the bacteria in colonizing the host and provide resistance against host-defense mechanisms.

Crystal structure and in silico studies of dihydrodipicolinate synthase (DHDPS) from Aquifex aeolicus.

Dihydrodipicolinate synthase (DHDPS, E.C.4.2.1.52) catalyzes the first committed step in the lysine biosynthetic pathway: the condensation of (S)-aspartate semialdehyde and pyruvate to form (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. Since (S)-lysine biosynthesis does not occur in animals, DHDPS is an attractive target for rational antibiotic and herbicide design. Here, we report the crystal structure of DHDPS from a hyperthermophilic bacterium Aquifex aeolicus (AqDHDPS). L-Lysine is used as an important animal feed additive where the production is at the level of 1.5 million tons per year. The biotechnological manufacture of lysine has been going for more than 50 years which includes over synthesis and reverse engineering of DHDPS. AqDHDPS revealed a unique disulfide linkage which is not conserved in the homologues of AqDHDPS. In silico mutation of C139A and intermolecular ion-pair residues and the subsequent molecular dynamics simulation of the mutants showed that these residues are critical for the stability of AqDHDPS tetramer. MD simulations of AqDHDPS at three different temperatures (303, 363 and 393 K) revealed that the molecule is stable at 363 K. Thus, this structural and in silico study of AqDHDPS likely provides additional details towards the rational and structure-based design of hyper-L-lysine producing bacterial strains.

Molecular dynamic simulations of the tubulin-human gamma synuclein complex: structural insight into the regulatory mechanism involved in inducing resistance against Taxol.

Members of the synuclein family (α, ß and γ synucleins) are intrinsically disordered in nature and play a crucial role in the progression of various neurodegenerative disorders and cancers. The association of γSyn with both BubR1 as well as microtubule subunits renders resistance against various anti-cancer drugs. However, the structural aspects underlying drug resistance have not been explored. In this study, the mechanism involved in the association between γSyn and microtubule subunits (αßTub) was investigated and the results reveal a strong interaction between γSyn and the tail regions of αßTub. Complexation of γSyn induces conformational rearrangements in the nucleotide binding loops (NBL), interdomain and tail regions of both α and ßTub. Moreover, in ßTub, the massive displacement observed in M and S loops significantly alters the binding site of microtubule targeting drugs like Taxol. The resulting weak association between Taxol and ßTub of the γSyn-αßTub complex was confirmed by molecular dynamic simulation studies. In addition, the effect of Taxol on NBL, M and S loops of αßTub, is reversed in the presence of γSyn. These results clearly indicate that the presence of γSyn annulled the allosteric regulation imposed by Taxol on the αßTub complex as well as preventing the binding of microtubule targeting drugs, which eventually leads to the development of resistance against these drugs in cancer cells.