Vivian Saridakis

Structural proteomics of an archaeon.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, ∼20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

Molecular recognition of p53 and MDM2 by USP7/HAUSP

The ubiquitin-specific protease, USP7, has key roles in the p53 pathway whereby it stabilizes both p53 and MDM2. We show that the N-terminal domain of USP7 binds two closely spaced 4-residue sites in both p53 and MDM2, falling between p53 residues 359–367 and MDM2 residues 147–159. Cocrystal structures with USP7 were determined for both p53 peptides and for one MDM2 peptide. These peptides bind the same surface of USP7 as Epstein-Barr nuclear antigen-1, explaining the competitive nature of the interactions. The structures and mutagenesis data indicate a preference for a P/AXXS motif in peptides that bind USP7. Contacts made by serine are identical and crucial for all peptides, and Trp165 in the peptide-binding pocket of USP7 is also crucial. These results help to elucidate the mechanism of substrate recognition by USP7 and the regulation of the p53 pathway.

Crystal structure of dTDP-4-keto-6-deoxy-D-hexulose 3,5-epimerase from Methanobacterium thermoautotrophicum complexed with dTDP.

Deoxythymidine diphosphate (dTDP)-4-keto-6-deoxy-d-hexulose 3,5-epimerase (RmlC) is involved in the biosynthesis of dTDP-l-rhamnose, which is an essential component of the bacterial cell wall. The crystal structure of RmlC from Methanobacterium thermoautotrophicumwas determined in the presence and absence of dTDP, a substrate analogue. RmlC is a homodimer comprising a central jelly roll motif, which extends in two directions into longer β-sheets. Binding of dTDP is stabilized by ionic interactions to the phosphate group and by a combination of ionic and hydrophobic interactions with the base. The active site, which is located in the center of the jelly roll, is formed by residues that are conserved in all known RmlC sequence homologues. The conservation of the active site residues suggests that the mechanism of action is also conserved and that the RmlC structure may be useful in guiding the design of antibacterial drugs.

The crystal structure of hypothetical protein MTH1491 from Methanobacterium thermoautotrophicum.

As part of our structural proteomics initiative, we have determined the crystal structure of MTH1491, a previously uncharacterized hypothetical protein from Methanobacterium thermoautotrophicum. MTH1491 is one of numerous structural genomics targets selected in a genome‐wide survey of uncharacterized proteins. It belongs to a family of proteins whose biological function is not known. The crystal structure of MTH1491, the first structure for this family of proteins, consists of an overall five‐stranded parallel β‐sheet with strand order 51234 and flanking helices. The oligomeric form of this molecule is a trimer as seen from both crystal contacts and gel filtration studies. Analysis revealed that the structure of MTH1491 is similar to that of dehydrogenases, amidohydrolases, and oxidoreductases. Using a combination of sequence and structural analyses, we showed that MTH1491 does not belong to either the dehydrogenase or the amidohydrolase superfamilies of proteins.

Crystal structure of Methanobacterium thermoautotrophicum conserved protein MTH1020 reveals an NTN-hydrolase fold.

Vivian Saridakis, Dinesh Christendat,* Anders Thygesen, Cheryl H. Arrowsmith, Aled M. Edwards, and Emil F. Pai Division of Molecular and Structural Biology, Ontario Cancer Institute, Toronto, Ontario, Canada Clinical Genomics Center, University Health Network, Toronto, Ontario, Canada Departments of Medical Biophysics, Biochemistry and Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada

Nicotinamide mononucleotide adenylyltransferase displays alternate binding modes for nicotinamide nucleotides

Two thirds of the clinically useful antibiotics are naturally produced in actinomycetes, especially in Streptomyces species. These antibiotics vary highly in their chemical structures, some examples being amphenicols (chloramphenicol), polyketides (tetracyclin) or aminoglycosides (streptomycin). Enzymes involved in the synthesis of aminoglycoside antibiotics (AGAs) are organized in large gene clusters containing 24 or more enzymes. The AGA family can be further divided into several subfamilies; the NEO subfamily takes the common precursor paromamine, which is further modified to AGAs such as neomycin, ribostamycin and lividomycin. [1] Lividomycin B and neomycin B members of the NEO subfamily are produced by enzymes of the LIV/NEO gene cluster. The aminotransferases that catalyze the terminal transamination reaction (LivB and NeoB respectively) utilise the cofactor pyridoxal-5’phosphate (PLP). LivB catalyzes the transamination reaction of 6’’’oxoparomomycin to the antibiotic paromomycin, which is also a precursor of lividomycin B, whereas NeoB performs the transamination of 6’’’-oxoneomycin C to neomycin C. [1] LivB and NeoB were expressed in Streptomyces sp., purified via Ni-affinity chromatography and crystallized. The structure of LivB could be solved using the “magic triangle compound” I3C [2] for SAD phasing, and that of NeoB by molecular replacement using the LivB structure as search model. Soaking of LivB crystals with the cofactor PLP, an amino donor and the end product paromomycin yielded crystal structures of the PLP-bound enzyme and the complex structure of LivB with an aldimine paromomycin-PLP intermediate. The latter represents a molecular snapshot of a key intermediate of the enzymatic reaction the transamination at the 6’’’ position of the lividomycin B. These structures provide a basis for analyzing substrate specificities of other carbohydrate-modifying aminotransferases.

Structural insight on the mechanism of regulation of the MarR family of proteins

How life emerged on Earth remains one of the great Mysteries for mankind. Molecules with backbones forming stable double helices, held by self-association, and capable of auto-replication and more precisely nucleotides held by Watson-Crick base pairings were considered as the seminal building blocks of life. Many scenarios involving extreme conditions were described, all of them dealing with the extreme pressure conditions of the “primary soup” that was present on earth at prebiotic stages. High-pressure molecular crystallography (HPMX) investigation of DNA was undertaken using crystals of the d(GGTATACC) octamer, in the range 0.2-2 GPa. This sequence crystallizes in the hexagonal P61 space group and is particularly interesting because it includes in a A-DNA crystal matrix, the B-form of DNA, leading us to simultaneously monitor the two forms of DNA under pressure. The 3D structure of d(GGTATACC) was recorded at ambient pressure, 0.55, 1.09 and 1.39 GPa and refined at 1.6 Å resolution. Fiber diagrams of the embedded B-DNA that superpose to the diffraction pattern of the A-DNA were analyzed from ambient pressure to up 1.9 GPa. A large axial compression of the DNA is observed (11 % at 1.39 GPa). The average base-step varies in A-DNA from 2.92 down to 2.73 Å, and in B-DNA from 3.40 to 3.10 Å. Surprisingly, in the case of A-DNA, the geometry of Watson-Crick base parings remains essentially invariant in the domain of pressure up to 1.39 GPa. Above 1.4 GPa, the crystal structure irreversibly deteriorates while the B-DNA fiber diagram still persists above 2 GPa. The remarkable stability and adaptation of d(GGTATACC) to high pressure is clearly associated with the base-paired double helix topology of the molecule by which it behaves as a molecular spring.