Saravanamuthu Thiyagarajan

Evolution of metal(loid) binding sites in transcriptional regulators.

Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals, and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II), and Ni(II). These homodimeric repressors bind to DNA in the absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, and the Cd(II) binding sites were in two different locations in two Cd(II)-responsive regulators. We hypothesize that ArsR/SmtB repressors have a common backbone structure, that of a winged helix DNA-binding protein, but have considerable plasticity in the location of inducer binding sites. Here we show that an As(III)-responsive member of the family, CgArsR1 from Corynebacterium glutamicum, binds As(III) to a cysteine triad composed of Cys15, Cys16, and Cys55. This binding site is clearly unrelated to the binding sites of other characterized ArsR/SmtB family members. This is consistent with our hypothesis that metal(loid) binding sites in DNA binding proteins evolve convergently in response to persistent environmental pressures.

Aquaglyceroporins: ancient channels for metalloids

The identification of aquaglyceroporins as uptake channels for arsenic and antimony shows how these toxic elements can enter the food chain, and suggests that food plants could be genetically modified to exclude arsenic while still accumulating boron and silicon.

Role of Bound Zn(II) in the CadC Cd(II)/Pb(II)/Zn(II)-responsive Repressor

The Staphylococcus aureus plasmid pI258 cadCA operon encodes a P-type ATPase, CadA, that confers resistance to Cd(II)/Pb(II)/Zn(II). Expression is regulated by CadC, a homodimeric repressor that dissociates from the cad operator/promoter upon binding of Cd(II), Pb(II), or Zn(II). CadC is a member of the ArsR/SmtB family of metalloregulatory proteins. The crystal structure of CadC shows two types of metal binding sites, termed Site 1 and Site 2, and the homodimer has two of each. Site 1 is the physiological inducer binding site. The two Site 2 metal binding sites are formed at the dimerization interface. Site 2 is not regulatory in CadC but is regulatory in the homologue SmtB. Here the role of each site was investigated by mutagenesis. Both sites bind either Cd(II) or Zn(II). However, Site 1 has higher affinity for Cd(II) over Zn(II), and Site 2 prefers Zn(II) over Cd(II). Site 2 is not required for either derepression or dimerization. The crystal structure of the wild type with bound Zn(II) and of a mutant lacking Site 2 was compared with the SmtB structure with and without bound Zn(II). We propose that an arginine residue allows for Zn(II) regulation in SmtB and, conversely, a glycine results in a lack of regulation by Zn(II) in CadC. We propose that a glycine residue was ancestral whether the repressor binds Zn(II) at a Site 2 like CadC or has no Site 2 like the paralogous ArsR and implies that acquisition of regulatory ability in SmtB was a more recent evolutionary event.

Regulatory Activities of Four ArsR Proteins in Agrobacterium tumefaciens 5A

ABSTRACT ArsR is a well-studied transcriptional repressor that regulates microbe-arsenic interactions. Most microorganisms have an arsR gene, but in cases where multiple copies exist, the respective roles or potential functional overlap have not been explored. We examined the repressors encoded by arsR1 and arsR2 (ars1 operon) and by arsR3 and arsR4 (ars2 operon) in Agrobacterium tumefaciens 5A. ArsR1 and ArsR4 are very similar in their primary sequences and diverge phylogenetically from ArsR2 and ArsR3, which are also quite similar to one another. Reporter constructs (lacZ) for arsR1, arsR2, and arsR4 were all inducible by As(III), but expression of arsR3 (monitored by reverse transcriptase PCR) was not influenced by As(III) and appeared to be linked transcriptionally to an upstream lysR-type gene. Experiments using a combination of deletion mutations and additional reporter assays illustrated that the encoded repressors (i) are not all autoregulatory as is typically known for ArsR proteins, (ii) exhibit variable control of each others encoding genes, and (iii) exert variable control of other genes previously shown to be under the control of ArsR1. Furthermore, ArsR2, ArsR3, and ArsR4 appear to have an activator-like function for some genes otherwise repressed by ArsR1, which deviates from the well-studied repressor role of ArsR proteins. The differential regulatory activities suggest a complex regulatory network not previously observed in ArsR studies. The results indicate that fine-scale ArsR sequence deviations of the reiterated regulatory proteins apparently translate to different regulatory roles. IMPORTANCE Given the significance of the ArsR repressor in regulating various aspects of microbe-arsenic interactions, it is important to assess potential regulatory overlap and/or interference when a microorganism carries multiple copies of arsR. This study explores this issue and shows that the four arsR genes in A. tumefaciens 5A, associated with two separate ars operons, encode proteins exhibiting various degrees of functional overlap with respect to autoregulation and cross-regulation, as well as control of other functional genes. In some cases, differences in regulatory activity are associated with only limited differences in protein primary structure. The experiments summarized herein also present evidence that ArsR proteins appear to have activator functions, representing novel regulatory activities for ArsR, previously known only to be a repressor.

Hexammineruthenium(III) ion interactions with Z-DNA

The hexamer duplex d(CGCGCA).d(TGCGCG) was crystallized with hexammineruthenium(III) ions in an orthorhombic space group; the crystals diffracted to 1.54 A resolution. Strong ion interactions with the adenine base induce a tautomeric shift from the amino to the imino form. Consequently, the A.T base pairing is disrupted. This structural study may be relevant to metal toxicity.

Unconventional actins and actin-binding proteins in human protozoan parasites.

Actin and its regulatory proteins play a key role in several essential cellular processes such as cell movement, intracellular trafficking and cytokinesis in most eukaryotes. While these proteins are highly conserved in higher eukaryotes, a number of unicellular eukaryotic organisms contain divergent forms of these proteins which have highly unusual biochemical and structural properties. Here, we review the biochemical and structural properties of these unconventional actins and their core binding proteins which are present in commonly occurring human protozoan parasites.

Identification of New Inhibitors for Human SIRT1: An in-silico Approach

BACKGROUND Human SIRT1 is a class III histone deacetylase (HDAC) family protein. As the overexpression of hSIRT1 leads to cancer, inhibiting its HDAC function may be a better strategy for the treatment of cancer. Till now, only a few reported inhibitor compounds have reached the stage of animal studies; hence, identifying high efficacy inhibitors of hSIRT1 is essential. OBJECTIVE The main objective of the study is to obtain a new class of inhibitor compounds of hSIRT1 by the rational structure-based method. METHODOLOGY We performed virtual screening using AutoDock Vina for the HDAC domain of hSIRT1 against the Drug- Bank library containing 1,716 compounds. The recently determined crystal structure of the HDAC domain of hSIRT1 (PDB Id: 4KXQ) was used for docking studies. Subsequently, we performed molecular dynamics simulations and an invitro deacetylase assay for selected compounds. RESULTS Virtual screening studies yielded seven compounds from two chemical classes, namely diphenyl and oxycoumarin derivatives. Molecular dynamic simulations confirmed that the predicted seven compounds bind well to their respective complex structures. Moreover, four commercially available drugs containing the predicted compounds showed significant inhibition of hSIRT1 deacetylase activity in comparison to the known hSIRT1 inhibitor (sirtinol). CONCLUSION Our results indicate that the compounds of the diphenyl and oxycoumarin series may serve as useful scaffolds in the development of new chemical libraries of hSIRT1 inhibitory activity.

Mutations in ARSB in MPS VI patients in India

Mucopolysaccharidosis VI (MPS VI) is an autosomal recessive inborn error of metabolism caused by mutations in the arylsulfatase B gene (ARSB) and consequent deficient activity of ARSB, a lysosomal enzyme. We present here the results of a study undertaken to identify the mutations in ARSB in MPS VI patients in India. Around 160 ARSB mutations, of which just 4 are from India, have been reported in the literature. Our study covered nine MPS VI patients from eight families. Both familial mutations were found in seven families, and only one mutation was found in one family. Seven mutations were found — four novel (p.G38_G40del3, p.C91R, p.L98R and p.R315P), two previously reported from India (p.D53N and p.W450C), and one reported from outside India (p.R160Q). One mutation, p.W450C, was present in two families, and the other six mutations were present in one family each. Analysis of the molecular structure of the enzyme revealed that most of these mutations either cause loss of an active site residue or destabilize the structure of the enzyme. The only previous study on mutations in ARSB in Indian MPS VI patients, by Kantaputra et al. 2014 [1], reported four novel mutations of which two (p.D53N and p.W450C) were found in our study as well. Till date, nine mutations have been reported from India, through our study and the Kantaputra study. Eight out of these nine mutations have been found only in India. This suggests that the population studied by us might have its own typical set of mutations, with other populations equally likely to have their own set of mutations.

Purification, crystallization and preliminary X-ray diffraction studies of the arsenic repressor ArsR from Corynebacterium glutamicum

ArsR is a member of the SmtB/ArsR family of metalloregulatory proteins that regulate prokaryotic arsenic-resistance operons. Here, the crystallization and preliminary X-ray diffraction studies of a cysteine-free derivative of ArsR from Corynebacterium glutamicum (CgArsR-C15/16/55S) are reported. CgArsR-C15/16/55S was expressed, purified, crystallized and X-ray diffraction data were collected to 1.86 Å resolution. The protein crystallized in a tetragonal space group (P4), with unit-cell parameters a = b = 41.84, c = 99.47 Å.

Sequence-dependent structural effects in left-handed DNA

About 25 years ago the DNA sequence d(CGCGCG)2 was crystallized, and the structure solved as a left-handed helix, dubbed Z-DNA. Many crystal structures of variants of this first sequence have since been obtained. In our laboratory we have focused on what happens to the helical structure, inter-helical interactions, and the solvent interactions when a single A:T (adenine–thymine) base pair replaces a G:C (guanine–cytosine) base pair in the hexameric sequence. We present here a review of these studies. The review addresses three topics. In the first part, it discusses the effect of A:T base pairs on the structure of Z-DNA. The crystal structures demonstrate that the degree of structural perturbation that an A:T base pair produces depends on its position in the sequence, as this alters the length of the G:C tract. The presence of four continuous G:C base pairs appears to be necessary and sufficient to nucleate a Z-type structure. In the second part, the review deals with interactions between helices and crystal packing modes in Z-DNA. It examines the consequences to these aspects, of the presence of an A:T base pair in the sequence. Hexameric duplexes may be approximated as cylinders. This leads to degeneracy in crystal packing modes in Z-DNA, particularly at low resolution. The presence of a single A:T base pair breaks the two-fold symmetry in the sequence and gives rise to directionality in the cylinders. This has interesting effects on the inter-helical interactions, and therefore on the crystal packing. Metal ion and solvent interactions are discussed in the third section of the review. Different ions have different modes of binding to the helices. Some of these are specific to the sequence and lead not only to subtle, but well-marked, variations in the helical structure, but also to differences in helical contacts and therefore crystal packing. In some cases they induce disorder in crystal packing. In others, the ions interact with the helices in a non-specific manner. Contents 1. Introduction 338 2. Sequence-dependent structure 339 3. Crystal packing 344 4. Solvent and ion interaction 350 Acknowledgements 353 References 353 Subject index 355