Farhad Forouhar

The structural basis of cyclic diguanylate signal transduction by PilZ domains.

The second messenger cyclic diguanylate (c‐di‐GMP) controls the transition between motile and sessile growth in eubacteria, but little is known about the proteins that sense its concentration. Bioinformatics analyses suggested that PilZ domains bind c‐di‐GMP and allosterically modulate effector pathways. We have determined a 1.9 Å crystal structure of c‐di‐GMP bound to VCA0042/PlzD, a PilZ domain‐containing protein from Vibrio cholerae. Either this protein or another specific PilZ domain‐containing protein is required for V. cholerae to efficiently infect mice. VCA0042/PlzD comprises a C‐terminal PilZ domain plus an N‐terminal domain with a similar β‐barrel fold. C‐di‐GMP contacts seven of the nine strongly conserved residues in the PilZ domain, including three in a seven‐residue long N‐terminal loop that undergoes a conformational switch as it wraps around c‐di‐GMP. This switch brings the PilZ domain into close apposition with the N‐terminal domain, forming a new allosteric interaction surface that spans these domains and the c‐di‐GMP at their interface. The very small size of the N‐terminal conformational switch is likely to explain the facile evolutionary diversification of the PilZ domain.

Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-Å resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate l-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the l-stereospecificity. A second, possibly allosteric, l-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygen-binding site that would also involve interactions with the ammonium moiety of l-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.

Crystal Structure of Pea Toc34 - a Novel Gtpase of the Chloroplast Protein Translocon

Toc34, a 34-kDa integral membrane protein, is a member of the Toc (translocon at the outer-envelope membrane of chloroplasts) complex, which associates with precursor proteins during protein transport across the chloroplast outer membrane. Here we report the 2.0 Å resolution crystal structure of the cytosolic part of pea Toc34 in complex with GDP and Mg2+. In the crystal, Toc34 molecules exist as dimers with features resembling those found in a small GTPase in complex with a GTPase activating protein (GAP). However, gel filtration experiments revealed that dimeric and monomeric forms of Toc34 coexisted in phosphate saline buffer solution at pH 7.2. Mutation of Arg 128, an essential residue for dimerization, to an Ala residue led to the formation of an exclusively monomeric species whose GTPase activity is significantly reduced compared to that of wild type Toc34. These results, together with a number of structural features unique to Toc34, suggest that each monomer acts as a GAP on the other interacting monomer.

Understanding the physical properties that control protein crystallization by analysis of large-scale experimental data.

Crystallization is the most serious bottleneck in high-throughput protein-structure determination by diffraction methods. We have used data mining of the large-scale experimental results of the Northeast Structural Genomics Consortium and experimental folding studies to characterize the biophysical properties that control protein crystallization. This analysis leads to the conclusion that crystallization propensity depends primarily on the prevalence of well-ordered surface epitopes capable of mediating interprotein interactions and is not strongly influenced by overall thermodynamic stability. We identify specific sequence features that correlate with crystallization propensity and that can be used to estimate the crystallization probability of a given construct. Analyses of entire predicted proteomes demonstrate substantial differences in the amino acid–sequence properties of human versus eubacterial proteins, which likely reflect differences in biophysical properties, including crystallization propensity. Our thermodynamic measurements do not generally support previous claims regarding correlations between sequence properties and protein stability.

Nicotinamide adenine dinucleotide metabolism as an attractive target for drug discovery

Nicotinamide adenine dinucleotide (NAD+) has crucial roles in many cellular processes, both as a coenzyme for redox reactions and as a substrate to donate ADP-ribose units. Enzymes involved in NAD+ metabolism are attractive targets for drug discovery against a variety of human diseases, including cancer, multiple sclerosis, neurodegeneration and Huntington’s disease. A small-molecule inhibitor of nicotinamide phosphoribosyltransferase, an enzyme in the salvage pathway of NAD+ biosynthesis, is presently in clinical trials against cancer. An analog of a kynurenine pathway intermediate is efficacious against multiple sclerosis in an animal model. Indoleamine 2,3-dioxygenase plays an important role in immune evasion by cancer cells and other disease processes. Inhibitors against kynurenine 3-hydroxylase can reduce the production of neurotoxic metabolites while increasing the production of neuroprotective compounds. This review summarizes the existing knowledge on NAD+ metabolic enzymes, with emphasis on their relevance for drug discovery.

Computational Design of Catalytic Dyads and Oxyanion Holes for Ester Hydrolysis

Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by side chains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (k(cat)/K(M)) of 400 M(-1) s(-1) for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.

Identification of Eukaryotic and Prokaryotic Methylthiotransferase for Biosynthesis of 2-Methylthio-N6-threonylcarbamoyladenosine in tRNA

Bacterial and eukaryotic transfer RNAs have been shown to contain hypermodified adenosine, 2-methylthio-N6-threonylcarbamoyladenosine, at position 37 (A37) adjacent to the 3′-end of the anticodon, which is essential for efficient and highly accurate protein translation by the ribosome. Using a combination of bioinformatic sequence analysis and in vivo assay coupled to HPLC/MS technique, we have identified, from distinct sequence signatures, two methylthiotransferase (MTTase) subfamilies, designated as MtaB in bacterial cells and e-MtaB in eukaryotic and archaeal cells. Both subfamilies are responsible for the transformation of N6-threonylcarbamoyladenosine into 2-methylthio-N6-threonylcarbamoyladenosine. Recently, a variant within the human CDKAL1 gene belonging to the e-MtaB subfamily was shown to predispose for type 2 diabetes. CDKAL1 is thus the first eukaryotic MTTase identified so far. Using purified preparations of Bacillus subtilis MtaB (YqeV), a CDKAL1 bacterial homolog, we demonstrate that YqeV/CDKAL1 enzymes, as the previously studied MTTases MiaB and RimO, contain two [4Fe-4S] clusters. This work lays the foundation for elucidating the function of CDKAL1.

Structural and Functional Evidence for Bacillus subtilis PaiA as a Novel N1-Spermidine/Spermine Acetyltransferase

Bacillus subtilis PaiA has been implicated in the negative control of sporulation as well as production of degradative enzymes. PaiA shares recognizable sequence homology with N-acetyltransferases, including those that can acetylate spermidine/spermine substrates. We have determined the crystal structure of PaiA in complex with CoA at 1.9 Å resolution and found that PaiA is a member of the N-acetyltransferase superfamily of enzymes. Unexpectedly, we observed the binding of an oxidized CoA dimer in the active site of PaiA, and the structural information suggests the substrates of the enzyme could be linear, positively charged compounds. Our biochemical characterization is also consistent with this possibility, since purified PaiA possesses N1-acetyltransferase activity toward polyamine substrates including spermidine and spermine. Further, conditional overexpression of PaiA in bacteria results in increased acetylation of endogenous spermidine pools. Thus, our structural and biochemical analyses indicate that PaiA is a novel N-acetyltransferase capable of acetylating both spermidine and spermine. In this way, the pai operon may function in regulating intracellular polyamine concentrations and/or binding capabilities. In addition to preventing toxicity due to polyamine excess, this function may also serve to regulate expression of certain bacterial gene products such as those involved in sporulation.

Use of a synthetic salicylic acid analog to investigate the roles of methyl salicylate and its esterases in plant disease resistance.

We previously demonstrated that salicylic acid-binding protein 2 (SABP2) of tobacco is an integral component of systemic acquired resistance (SAR). SABP2 is a methyl salicylate (MeSA) esterase that has high affinity for SA, which feedback inhibits its esterase activity. MeSA esterase activity is required in distal, healthy tissue of pathogen-infected plants to hydrolyze MeSA, which functions as a long-distance, phloem-mobile SAR signal; this hydrolysis releases the biologically active defense hormone SA. In this study, we examined the inhibitory interaction of SA with SABP2, and identified a synthetic SA analog, 2,2,2,2′-tetra-f luoroacetophenone (tetraFA) that, like SA, competitively inhibits the activity of SABP2 and targets esterases, which utilize MeSA as a substrate. However, in contrast to SA, tetraFA does not induce downstream defense responses and, therefore, is effective in planta at blocking SAR development in tobacco mosaic virus (TMV)-infected tobacco and Pseudomonas syringae-infected Arabidopsis. These results confirm the importance of SABP2 and MeSA for SAR development in tobacco and establish similar roles for MeSA and the orthologs of SABP2 in Arabidopsis. Moreover, they demonstrate that tetraFA can be used to determine whether MeSA and its corresponding esterase(s) play a role in SAR signaling in other plant species. In planta analyses using tetraFA, in conjunction with leaf detachment assays and MeSA quantification, were used to assess the kinetics with which MeSA is generated in pathogen-infected leaves, transmitted through the phloem, and processed in the distal healthy leaves. In TMV-infected tobacco, these studies revealed that critical amounts of MeSA are generated, transmitted, and processed between 48 and 72 h post primary infection.

Two Fe-S clusters catalyze sulfur insertion by radical-SAM methylthiotransferases

How living organisms create carbon-sulfur bonds during the biosynthesis of critical sulfur-containing compounds is still poorly understood. The methylthiotransferases MiaB and RimO catalyze sulfur insertion into tRNAs and ribosomal protein S12, respectively. Both belong to a subgroup of radical-S-adenosylmethionine (radical-SAM) enzymes that bear two [4Fe-4S] clusters. One cluster binds S-adenosylmethionine and generates an Ado• radical via a well-established mechanism. However, the precise role of the second cluster is unclear. For some sulfur-inserting radical-SAM enzymes, this cluster has been proposed to act as a sacrificial source of sulfur for the reaction. In this paper, we report parallel enzymological, spectroscopic and crystallographic investigations of RimO and MiaB, which provide what is to our knowledge the first evidence that these enzymes are true catalysts and support a new sulfation mechanism involving activation of an exogenous sulfur cosubstrate at an exchangeable coordination site on the second cluster, which remains intact during the reaction.