Domain Structure of Virulence-associated Response Regulator PhoP of Mycobacterium tuberculosis: ROLE OF THE LINKER REGION IN REGULATOR-PROMOTER INTERACTION(S)

The PhoP and PhoR proteins from Mycobacterium tuberculosis form a highly specific two-component system that controls expression of genes involved in complex lipid biosynthesis and regulation of unknown virulence determinants. The several functions of PhoP are apportioned between a C-terminal effector domain (PhoPC) and an N-terminal receiver domain (PhoPN), phosphorylation of which regulates activation of the effector domain. Here we show that PhoPN, on its own, demonstrates PhoR-dependent phosphorylation. PhoPC, the truncated variant bearing the DNA binding domain, binds in vitro to the target site with affinity similar to that of the full-length protein. To complement the finding that residues spanning Met¹ to Arg¹³⁸ of PhoP constitute the minimal functional PhoPN, we identified Arg¹⁵⁰ as the first residue of the distal PhoPC domain capable of DNA binding on its own, thereby identifying an interdomain linker. However, coupling of two functional domains together in a single polypeptide chain is essential for phosphorylation-coupled DNA binding by PhoP. We discuss consequences of tethering of two domains on DNA binding and demonstrate that linker length and not individual residues of the newly identified linker plays a critical role in regulating interdomain interactions. Together, these results have implications for the molecular mechanism of transmission of conformation change associated with phosphorylation of PhoP that results in the altered DNA recognition by the C-terminal domain.     

Switching Protein-DNA Recognition Specificity by Single-Amino-Acid Substitutions in the P1 par Family of Plasmid Partition Elements

The P1, P7, and pMT1 par systems are members of the P1 par family of plasmid partition elements. Each has a ParA ATPase and a ParB protein that recognizes the parS partition site of its own plasmid type to promote the active segregation of the plasmid DNA to daughter cells. ParB contacts two parS motifs known as BoxA and BoxB, the latter of which determines species specificity. We found that the substitution of a single orthologous amino acid in ParB for that of a different species has major effects on the specificity of recognition. A single change in ParB can cause a complete switch in recognition specificity to that of another species or can abolish specificity. Specificity changes do not necessarily correlate with changes in the gross DNA binding properties of the protein. Molecular modeling suggests that species specificity is determined by the capacity to form a hydrogen bond between ParB residue 288 and the second base in the BoxB sequence. As changes in just one ParB residue and one BoxB base can alter species specificity, plasmids may use such simple changes to evolve new species rapidly.     

Thermodynamic and Structural Basis for Relaxation of Specificity in Protein–DNA Recognition

As a novel approach to the structural and functional properties that give rise to extremely stringent sequence specificity in protein–DNA interactions, we have exploited “promiscuous” mutants of EcoRI endonuclease to study the detailed mechanism by which changes in a protein can relax specificity. The A138T promiscuous mutant protein binds more tightly to the cognate GAATTC site than does wild-type EcoRI yet displays relaxed specificity deriving from tighter binding and faster cleavage at EcoRI* sites (one incorrect base pair). AAATTC EcoRI* sites are cleaved by A138T up to 170-fold faster than by wild-type enzyme if the site is abutted by a 5′-purine-pyrimidine (5′-RY) motif. When wild-type protein binds to an EcoRI* site, it forms structurally adapted complexes with thermodynamic parameters of binding that differ markedly from those of specific complexes. By contrast, we show that A138T complexes with 5′-RY-flanked AAATTC sites are virtually indistinguishable from wild-type-specific complexes with respect to the heat capacity change upon binding (?C°_P), the change in excluded macromolecular volume upon association, and contacts to the phosphate backbone. While the preference for the 5′-RY motif implicates contacts to flanking bases as important for relaxed specificity, local effects are not sufficient to explain the large differences in ?C°_P and excluded volume, as these parameters report on global features of the complex. Our findings therefore support the view that specificity does not derive from the additive effects of individual interactions but rather from a set of cooperative events that are uniquely associated with specific recognition.     

Changing the target base specificity of the EcoRV DNA methyltransferase by rational de novo protein-design

The EcoRV DNA-(adenine-N⁶)-methyltransferase (M.EcoRV) specifically modifies the first adenine residue within GATATC sequences. During catalysis, the enzyme flips its target base out of the DNA helix and binds it into a target base binding pocket which is formed in part by Lys16 and Tyr196. A cytosine residue is accepted by wild-type M.EcoRV as a substrate at a 31-fold reduced efficiency with respect to the k_cat/K_M values if it is located in a CT mismatch substrate (GCTATC/GATATC). Cytosine residues positioned in a CG base pair (GCTATC/GATAGC) are modified at much more reduced rates, because flipping out the target base is much more difficult in this case. We intended to change the target base specificity of M.EcoRV from adenine-N⁶ to cytosine-N⁴. To this end we generated, purified and characterized 15 variants of the enzyme, containing single, double and triple amino acid exchanges following different design approaches. One concept was to reduce the size of the target base binding pocket by site-directed mutagenesis. The K16R variant showed an altered specificity, with a 22-fold preference for cytosine as the target base in a mismatch substrate. This corresponds to a 680-fold change in specificity, which was accompanied by only a small loss in catalytic activity with the cytosine substrate. The K16R/Y196W variant no longer methylated adenine residues at all and its activity towards cytosine was reduced only 17-fold. Therefore, we have changed the target base specificity of M.EcoRV from adenine to cytosine by rational protein design. Because there are no natural paragons for the variants described here, a change of the target base specificity of a DNA interacting enzyme was possible by rational de novo design of its active site.     

Structure and function of the transketolase from Mycobacterium tuberculosis and comparison with the human enzyme

The transketolase (TKT) enzyme in Mycobacterium tuberculosis represents a novel drug target for tuberculosis treatment and has low homology with the orthologous human enzyme. Here, we report on the structural and kinetic characterization of the transketolase from M. tuberculosis (TBTKT), a homodimer whose monomers each comprise 700 amino acids. We show that TBTKT catalyses the oxidation of donor sugars xylulose-5-phosphate and fructose-6-phosphate as well as the reduction of the acceptor sugar ribose-5-phosphate. An invariant residue of the TKT consensus sequence required for thiamine cofactor binding is mutated in TBTKT; yet its catalytic activities are unaffected, and the 2.5 Å resolution structure of full-length TBTKT provides an explanation for this. Key structural differences between the human and mycobacterial TKT enzymes that impact both substrate and cofactor recognition and binding were uncovered. These changes explain the kinetic differences between TBTKT and its human counterpart, and their differential inhibition by small molecules. The availability of a detailed structural model of TBTKT will enable differences between human and M. tuberculosis TKT structures to be exploited to design selective inhibitors with potential antitubercular activity.     

A point mutation in the two-component regulator PhoP-PhoR accounts for the absence of polyketide-derived acyltrehaloses but not that of phthiocerol dimycocerosates in Mycobacterium tuberculosis H37Ra

Similarities between Mycobacterium tuberculosis phoP-phoR mutants and the attenuated laboratory strain M. tuberculosis H37Ra in terms of morphological and cytochemical properties, lipid content, gene expression and virulence attenuation prompted us to analyze the functionality of this two-component regulator in the latter strain. Sequence analysis revealed a base substitution resulting in a one-amino-acid change in the likely DNA-binding region of PhoP in H37Ra relative to H37Rv. Using gel-shift assays, we show that this mutation abrogates the ability of the H37Ra PhoP protein to bind to a 40-bp segment of its own promoter. Consistent with this result, the phoP gene from H37Rv but not that from H37Ra was able to restore the synthesis of sulfolipids, diacyltrehaloses and polyacyltrehaloses in an isogenic phoP-phoR knock-out mutant of M. tuberculosis Moreover, complementation of H37Ra with phoP from H37Rv fully restored sulfolipid, diacyltrehalose and polyacyltrehalose synthesis, clearly indicating that the lack of production of these lipids in H37Ra is solely due to the point mutation in phoP. Using a pks2-3/4 knock-out mutant of M. tuberculosis H37Rv, evidence is further provided that the above-mentioned polyketide-derived acyltrehaloses do not significantly contribute to the virulence of the tubercle bacillus in a mouse model of infection. Reasons for the attenuation of H37Ra thus most likely stand in other virulence factors, many of which are expected to belong to the PhoP regulon and another of which, unrelated to PhoP, appears to be the lack of production of phthiocerol dimycocerosates in this strain.     

Identification of important chemical groups of the hut mRNA for HutP interactions that regulate the hut operon in Bacillus subtilis

HutP is an RNA binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus species by binding to cis-acting regulatory sequences on hut mRNA. We recently solved the HutP crystal structure, which revealed a novel fold where three dimers are arranged in a 3-fold axis to form the hexamer. We also identified a minimal RNA binding element sufficient for HutP binding: three UAG trinucleotide motifs, each separated by 4 nt, located just upstream of the terminator. In the present study we have identified important RNA chemical groups essential for HutP interactions, by combining an in vitro selection strategy and analyses by site-specific base substitutions. These analyses suggest that each HutP molecule recognizes one UAG motif, where the first base (U) can be substituted with other bases, while the second and third bases (A and G) are required for the interactions. Further analyses of the chemical groups of the A and G bases in the UAG motif by modified base analogs suggested the importance of the exocyclic NH₂ group in these bases. Also, in this motif, only the 2′-OH group of A is important for HutP recognition. Considering the important chemical groups identified here, as well as the electrostatic potential analysis of HutP, we propose that Glu137 is one of the important residues for the HutP–RNA interactions.     

The Phe-X-Glu DNA binding motif of MutS. The role of hydrogen bonding in mismatch recognition

The crystal structures of MutS protein from Thermus aquaticus and Escherichia coli in a complex with a mismatch-containing DNA duplex reveal that the Glu residue in a conserved Phe-X-Glu motif participates in a hydrogen-bonded contact with either an unpaired thymidine or the thymidine of a G-T base-base mismatch. Here, the role of hydrogen bonding in mismatch recognition by MutS is assessed. The relative affinities of MutS for DNA duplexes containing nonpolar shape mimics of A and T, 4-methylbenzimidazole (Z), and difluorotoluene (F), respectively, that lack hydrogen bonding donors and acceptors, are determined in gel mobility shift assays. The results provide support for an induced fit mode of mismatch binding in which duplexes destabilized by mismatches are preferred substrates for kinking by MutS. Hydrogen bonding between the O epsilon 2 group of Glu and the mismatched base contributes only marginally to mismatch recognition and is significantly less important than the aromatic ring stack with the conserved Phe residue. A MutS protein in which Ala is substituted for Glu(38) is shown to be defective for mismatch repair in vivo. DNA binding studies reveal a novel role for the conserved Glu residue in the establishment of mismatch discrimination by MutS.     

Evolution of I-SceI homing endonucleases with increased DNA recognition site specificity

Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G₊₄ base pair for the wild-type A:T₊₄ base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T₊₄ were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T₊₄ or the C:G₊₄ base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G₊₄ recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T₊₄ target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G₊₄ target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed ∼36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G₊₄ substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.     

L. donovani XPRT: Molecular characterization and evaluation of inhibitors

Among all PRT enzymes of purine salvage pathway in Leishmania, XPRT (Xanthine phosphoribosyl transferase) is unique in its substrate specificity and their non-existence in human. It is an interesting protein not only for drug designing but also to understand the molecular determinants of its substrate specificity. Analysis of the 3D model of L. donovani XPRT (Ld-XPRT) revealed that Ile 209, Glu 215 and Tyr 208 may be responsible for the altered substrate specificity of Ld-XPRT. Comparisons with it's nearest homologue in humans, revealed significant differences between the two. A 28 residue long unique motif was identified in Ld-XPRT, which showed highest fluctuation upon substrate binding during MD simulations. In kinetic analysis, Ld-XPRT could phosphoribosylate xanthine, hypoxanthine and guanine with K_m values of 7.27, 8.13, 8.48 μM and k_cat values of 2.24, 1.82, 1.19 min^− 1 respectively. Out of 159 compounds from docking studies, six compounds were characterized further by fluorescence spectroscopy, CD spectroscopy and enzyme inhibition studies. Fluorescence quenching experiment was performed to study the binding of inhibitors with Ld-XPRT and dissociation constants were calculated. Four compounds are bi-substrate analogues and show competitive inhibition with both the substrates (Xanthine and PRPP) of Ld-XPRT. The CD spectral analysis revealed that the binding of inhibitors to Ld-XPRT induce change in its tertiary structure, where as its secondary structure pattern remains unchanged. Two Ld-XPRT inhibitors (dGDP and cGMP), which also have ability to inhibit Leishmanial HGPRT, are predicted as potential drug candidates as it can inhibit both the important enzymes of the purine salvage pathway.