Inhibition of Tyrosine Phosphorylation of Sperm Flagellar Proteins, Outer Dense Fiber Protein-2 and Tektin-2, Is Associated With Impaired Motility During Capacitation of Hamster Spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

Mitochondrial and nuclear markers suggest Hanuman langur (Primates: Colobinae) polyphyly: Implications for their species status

Recent molecular studies on langurs of the Indian subcontinent suggest that the widely-distributed and morphologically variable Hanuman langurs (Semnopithecus entellus) are polyphyletic with respect to Nilgiri and urple-faced langurs. To further investigate this scenario, we have analyzed additional sequences of mitochondrial cytochrome b as well as nuclear protamine P1 genes from these species. The results confirm Hanuman langur polyphyly in the mitochondrial tree and the nuclear markers suggest that the Hanuman langurs share protamine P1 alleles with Nilgiri and purple-faced langurs. We recommend provisional splitting of the so-called Hanuman langurs into three species such that the taxonomy is consistent with their evolutionary relationships.

Molecular characterisation of ABC transporter type FtsE and FtsX proteins of Mycobacterium tuberculosis.

Elicitation of drug resistance and various survival strategies inside host macrophages have been the hallmarks of Mycobacterium tuberculosis as a successful pathogen. ATP Binding Cassette (ABC) transporter type proteins are known to be involved in the efflux of drugs in bacterial and mammalian systems. FtsE, an ABC transporter type protein, in association with the integral membrane protein FtsX, is involved in the assembly of potassium ion transport proteins and probably of cell division proteins as well, both of which being relevant to tubercle bacillus. In this study, we cloned ftsE gene of M. tuberculosis, overexpressed and purified. The recombinant MtFtsE-6xHis protein and the native MtFtsE protein were found localized on the membrane of E. coli and M. tuberculosis cells, respectively. MtFtsE-6xHis protein showed ATP binding in vitro, for which the K42 residue in the Walker A motif was found essential. While MtFtsE-6xHis protein could partially complement growth defect of E. coli ftsE temperature-sensitive strain MFT1181, co-expression of MtFtsE and MtFtsX efficiently complemented the growth defect, indicating that the MtFtsE and MtFtsX proteins might be performing an associated function. MtFtsE and MtFtsX-6xHis proteins were found to exist as a complex on the membrane of E. coli cells co-expressing the two proteins.

Characterization of single-stranded DNA-binding proteins from Mycobacteria. The carboxyl-terminal of domain of SSB is essential for stable association with its cognate RecA protein

Single-stranded DNA-binding proteins (SSB) play an important role in most aspects of DNA metabolism including DNA replication, repair, and recombination. We report here the identification and characterization of SSB proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis. Sequence comparison of M. smegmatis SSB revealed that it is homologous to M. tuberculosis SSB, except for a small spacer connecting the larger amino-terminal domain with the extreme carboxyl-terminal tail. The purified SSB proteins of mycobacteria bound single-stranded DNA with high affinity, and the association and dissociation constants were similar to that of the prototype SSB. The proteolytic signatures of free and bound forms of SSB proteins disclosed that DNA binding was associated with structural changes at the carboxyl-terminal domain. Significantly, SSB proteins from mycobacteria displayed high affinity for cognate RecA, whereas Escherichia coli SSB did not under comparable experimental conditions. Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteins in vivo. The carboxyl-terminal domain of M. smegmatis SSB, which is not essential for interaction with ssDNA, is the site of binding of its cognate RecA. These studies provide the first evidence for stable association of eubacterial SSB proteins with their cognate RecA, suggesting that these two proteins might function together during DNA repair and/or recombination.

Phage lambda beta protein, a component of general recombination, is associated with host ribosomal S1 protein

We have purified phage lambda beta protein produced by a recombinant plasmid carrying bet gene and confirm that it forms a complex with a protein of relative molecular mass 70 kDa. Therefore, beta protein, a component of general genetic recombination, is associated with two functionally diverse complexes; one containing exonuclease and the other 70 kDa protein. Using a number of independent methods, we show that 70 kDa protein is the ribosomal S1 protein of E. coli. Further, the association of 70 kDa protein with beta protein is biologically significant, as the former inhibits joining of the terminal ends of lambda chromosome and renaturation of complementary single stranded DNA promoted by the latter. More importantly, these findings initiate an understanding of an important mode of host- virus interaction in general with specific implication(s) in homologous genetic recombination.

A Single Mammalian Mitochondrial Translation Initiation Factor Functionally Replaces Two Bacterial Factors

The mechanism of translation in eubacteria and organelles is thought to be similar. In eubacteria, the three initiation factors IF1, IF2, and IF3 are vital. Although the homologs of IF2 and IF3 are found in mammalian mitochondria, an IF1 homolog has never been detected. Here, we show that bovine mitochondrial IF2 (IF2mt) complements E. coli containing a deletion of the IF2 gene (E. coli ΔinfB). We find that IF1 is no longer essential in an IF2mt-supported E. coli ΔinfB strain. Furthermore, biochemical and molecular modeling data show that a conserved insertion of 37 amino acids in the IF2mt substitutes for the function of IF1. Deletion of this insertion from IF2mt supports E. coli for the essential function of IF2. However, in this background, IF1 remains essential. These observations provide strong evidence that a single factor (IF2mt) in mammalian mitochondria performs the functions of two eubacterial factors, IF1 and IF2.

Evolutionary Significance of Self-acylation Property in Acyl Carrier Proteins

Acyl carrier protein is an integral component of many cellular metabolic processes. A number of studies have reported self-acylation behavior in acyl carrier proteins. Although AM exhibit high levels of similarity in their primary and tertiary structures, self-acylation behavior is restricted to only some ACPs that can be classified into two major families based on their function. The first family of ACPs is involved in polyketide biosynthesis, whereas the second family participates in fatty acid synthesis. Facilitated by the growing number of genome sequences available for analyses, large-scale phylogenetic studies were used in these studies to uncover as to how self-acylation behavior of acyl carrier proteins is linked with the evolution of metabolic pathways in organisms. These studies show that self-acylation behavior in acyl carrier proteins was lost during the course of evolution, with certain organisms and organelles viz. plastids, retaining it for specified functions. (C) 2009 IUBMB IUBMB Life, 61(8): 853-859, 2009

Inter-helical Interactions in Membrane Proteins: Analysis Based on the Local Backbone Geometry and the Side Chain Interactions

The availability of a significant number of the Structures of helical membrane proteins has prompted us to investigate the mode of helix-helix packing. In the present study, we have considered a dataset of alpha-helical membrane proteins representing Structures solved from all the known superfamilies. We have described the geometry of all the helical residues in terms of local coordinate axis at the backbone level. Significant inter-helical interactions have been considered as contacts by weighing the number of atom-atom contacts, including all the side-chain atoms. Such a definition of local axis and the contact criterion has allowed us to investigate the inter-helical interaction in a systematic and quantitative manner. We show that a single parameter (designated as alpha), which is derived from the parameters representing the Mutual orientation of local axes, is able to accurately Capture the details of helix-helix interaction. The analysis has been carried Out by dividing the dataset into parallel, anti-parallel, and perpendicular orientation of helices. The study indicates that a specific range of alpha value is preferred for interactions among the anti-parallel helices. Such a preference is also seen among interacting residues of parallel helices, however to a lesser extent. No such preference is seen in the case of perpendicular helices, the contacts that arise mainly due to the interaction Of Surface helices with the end of the trans-membrane helices. The Study Supports the prevailing view that the anti-parallel helices are well packed. However, the interactions between helices of parallel orientation are non-trivial. The packing in alpha-helical membrane proteins, which is systematically and rigorously investigated in this study, may prove to be useful in modeling of helical membrane proteins.

Identification and thermodynamic characterization of molten globule states of periplasmic binding proteins

Molten globule-like intermediates have been shown to occur during protein folding and are thought to be involved in protein translocation and membrane insertion. However, the determinants of molten globule stability and the extent of specific packing in molten globules is currently unclear. Using far- and near-UV CD and intrinsic and ANS fluorescence, we show that four periplasmic binding proteins (LBP, LIVBP, MBP, and RBP) form molten globules at acidic pH values ranging from 3.0 to 3.4. Only two of these (LBP and LIVBP) have similar sequences, but all four proteins adopt similar three-dimensional structures. We found that each of the four molten globules binds to its corresponding ligand without conversion to the native state. Ligand binding affinity measured by isothermal titration calorimetry for the molten globule state of LIVBP was found to be comparable to that of the corresponding native state, whereas for LBP, MBP, and RBP, the molten globules bound ligand with approximately 5-30-fold lower affinity than the corresponding native states. All four molten globule states exhibited cooperative thermal unfolding assayed by DSC. Estimated values of Delta C-p of unfolding show that these molten globule states contain 28-67% of buried surface area relative to the native states. The data suggest that molten globules of these periplasmic binding proteins retain a considerable degree of long range order. The ability of these sequentially unrelated proteins to form highly ordered molten globules may be related to their large size as well as an intrinsic property of periplasmic binding protein folds.

Amino acid interaction preferences in proteins

Understanding the key factors that influence the interaction preferences of amino acids in the folding of proteins have remained a challenge. Here we present a knowledge-based approach for determining the effective interactions between amino acids based on amino acid type, their secondary structure, and the contact based environment that they find themselves in the native state structure as measured by their number of neighbors. We find that the optimal information is approximately encoded in a 60 x 60 matrix describing the 20 types of amino acids in three distinct secondary structures (helix, beta strand, and loop). We carry out a clustering scheme to understand the similarity between these interactions and to elucidate a nonredundant set. We demonstrate that the inferred energy parameters can be used for assessing the fit of a given sequence into a putative native state structure.

How Effective Is The Data On Co-Occurrence Of Domains In Multi-Domain Proteins In Prediction Of Protein-Protein Interactions?

We explore the fuse of information on co-occurrence of domains in multi-domain proteins in predicting protein-protein interactions. The basic premise of our work is the assumption that domains co-occurring in a polypeptide chain undergo either structural or functional interactions among themselves. In this study we use a template dataset of domains in multidomain proteins and predict protein-protein interactions in a target organism. We note that maximum number of correct predictions of interacting protein domain families (158) is made in S. cerevisiae when the dataset of closely related organisms is used as the template followed by the more diverse dataset of bacterial proteins (48) and a dataset of randomly chosen proteins (23). We conclude that use of multi-domain information from organisms closely-related to the target can aid prediction of interacting protein families.

Role of 16S ribosomal RNA methylations in translation initiation in Escherichia coli

Translation initiation from the ribosomal P-site is the specialty of the initiator tRNAs (tRNA(fMet)). Presence of the three consecutive G-C base pairs (G29-C41, G30-C40 and G31-C39) in their anticodon stems, a highly conserved feature of the initiator tRNAs across the three kingdoms of life, has been implicated in their preferential binding to the P-site. How this feature is exploited by ribosomes has remained unclear. Using a genetic screen, we have isolated an Escherichia coli strain, carrying a G122D mutation in folD, which allows initiation with the tRNA(fMet) containing mutations in one, two or all the three G-C base pairs. The strain shows a severe deficiency of methionine and S-adenosylmethionine, and lacks nucleoside methylations in rRNA. Targeted mutations in the methyltransferase genes have revealed a connection between the rRNA modifications and the fundamental process of the initiator tRNA selection by the ribosome.

Rotavirus nonstructural proteins: a structural perspective

Rotavirus is a major cause of acute infantile diarrhoea worldwide. The virus genome consists of 11 segments of double-stranded RNA that codesfor six structural proteins (VP1-6) and six non-structural proteins(NSP1-6). NSPs are proteins expressed from the virus genome in the infected cell, but are not incorporated into the mature virus article. NSPs play an essential role in virus replication, morphogenesis and pathogenesis, and most of them exhibit multifunctional properties. Structure-function analysis of the NSPs is essential for understanding the molecular mechanisms by which the virus circumvents host innate immune responses, inhibits cellular protein synthesis, hijacks the protein synthetic machinery for its own propagation and manifests the disease process. Because of their essential roles in virus biology, NSPs represent potential targets for the development of antiviral agents. Determination of the three-dimensional structure of NSPs has been hindered due to low-level expression and aggregation. To date, the complete three-dimensional structure of only NSP2 has been determined. The structures of the N- and C-terminal domains of NSP3 and the diarrhoea-inducing domain of NSP4 have also been determined. This review primarily covers the structural and biological functions of the NSPs whose three-dimensional structural aspects have been fully or partially understood, but provides a brief account of other NSPs and the structural features of the mature virion as determined by electron cryomicroscopy.

Effects of membrane channel-forming polypeptides on mitochondrial oxidative phosphorylation. A comparison of alamethicin, gramicidin A, melittin and tetraacetyl melittin

Transmembrane channel-forming polypeptides can function as uncouplers of mitochondrial oxidative phosphorylation. The observed effects are dependent on the phosphate ion (Pi) concentration in the medium. At low Pi (2.5 mM) the order of uncoupling efficiencies is gramicidin A much greater than alamethicin greater than tetraacetyl melittin greater than melittin. The remarkably high activity of gramicidin A suggests insertion of preformed channel dimers into the membrane. It is also suggested that lipid phase association of peptides is necessary in the other cases. At Pi = 100 mM inhibitory effects are observed for alamethicin and tetraacetyl melittin. Less pronounced inhibition is seen for melittin, while no such effect is noted for gramicidin A. The site of inhibition is shown to be complex IV, and the differences in the behavior of the peptides are rationalized in terms of channel structures.