Dmitriy Alexeev

A novel protein-mineral interface

Transferrins transport Fe3+ and other metal ions in mononuclear-binding sites. We present the first evidence that a member of the transferrin superfamily is able to recognize multi-nuclear oxo-metal clusters, small mineral fragments that are the most abundant forms of many metals in the environment. We show that the ferric ion–binding protein from Neisseria gonorrhoeae (nFbp) readily binds clusters of Fe3+, Ti4+, Zr4+ or Hf4+ in solution. The 1.7 Å resolution crystal structure of Hf–nFbp reveals three distinct types of clusters in an open, positively charged cleft between two hinged protein domains. A di-tyrosyl cluster nucleation motif (Tyr195-Tyr196) is situated at the bottom of this cleft and binds either a trinuclear oxo-Hf cluster, which is capped by phosphate, or a pentanuclear cluster, which in turn can be capped with phosphate. This first high-resolution structure of a protein–mineral interface suggests a novel metal-uptake mechanism and provides a model for protein-mediated mineralization/dissimilation, which plays a critical role in geochemical processes.NOTE: In the version of this article initially published online, the institution affiliations were assigned incorrectly because of a mistake that occurred during production. The correct affiliations for all authors are as follows: Dmitriy Alexeev1, Haizhong Zhu2, Maolin Guo2,3, Weiqing Zhong2,4, Dominic J.B. Hunter2, Weiping Yang2,3, Dominic J. Campopiano2 and Peter J. Sadler2. All of the footnotes (corrected) are as follows: 1Institute of Cell and Molecular Biology, Michael Swann Building, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK; 2School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK; 3Current address: Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, USA; and 4Current address: School of Pharmacy, Second Military Medicine University, Shanghai 200433, China. We apologize for any inconvenience this may have caused. This mistake has been corrected in the HTML and print version of the article.

Mechanistic implications and family relationships from the structure of dethiobiotin synthetase.

BACKGROUND Biotin is the vitamin essential for many biological carboxylation reactions, such as the conversion of acetyl-coenzyme A (CoA) to malonyl-CoA in fatty acid biosynthesis. Dethiobiotin synthetase (DTBS) facilitates the penultimate, ureido ring closure in biotin synthesis, which is a non-biotin-dependent carboxylation. DTBS displays no sequence similarity to any other protein in the database. Structural studies provide a molecular insight into the reaction mechanism of DTBS. RESULTS We present the structure of DTBS refined to 1.80 A resolution with an R-factor of 17.2% for all terms plus unrefined data on the binding of the substrate, 7,8-diaminopelargonic acid and the product, dethiobiotin. These studies confirm that the protein forms a homodimer with each subunit folded as a single globular alpha/beta domain. The presence of sulphate ions in the crystals and comparisons with the related Ha-ras-p21 oncogene product are used to infer the ATP-binding site, corroborated by the difference electron density for the ATP analogue AMP-PNP. CONCLUSIONS This study establishes that the enzyme active site is situated at the dimer interface, with the substrate binding to one monomer and ATP to the other. The overall fold of DTBS closely resembles that of three other enzymes, adenylosuccinate synthetase (purA), Ha-ras-p21, and nitrogenase iron protein, that are unrelated by sequence or function, indicating that DTBS is a member of a diverse family of enzymes.

Synthesis, Structural and Biological Studies of Ubiquitin Mutants Containing (2S, 4S)-5-Fluoroleucine Residues Strategically Placed in the Hydrophobic Core

A fluoroleucine mutant of ubiquitin (cyan) with two strategically placed fluorine reporter groups in its core was synthesised. 1H NMR, crystallography, CD spectroscopy, biological assay and calorimetry indicate minimal structural and functional perturbation compared to the native protein (yellow; see picture). 19F NMR spectra indicate changes within the core during folding and unfolding.

Substrate binding and carboxylation by dethiobiotin synthetase — a kinetic and X-ray study

BACKGROUND The vitamin biotin is a ubiquitous prosthetic group of carboxylase and transcarboxylase enzymes. Biotin biosynthesis occurs by similar pathways in microorganisms and plants. The penultimate step in biotin biosynthesis, catalyzed by dethiobiotin synthetase (DTBS), involves a unique ATP-dependent N-carboxylation, resulting in formation of the ureido ring function of dethiobiotin. The first two steps of dethiobiotin formation, which is a complex, multistep enzymatic reaction, have been elucidated by a combination of X-ray crystallography and kinetic methods. RESULTS The first step in catalysis by DTBS is the formation of an enzyme-substrate complex and the second is the enzymatic carboxylation of the bound substrate. Both steps are Mg2+ dependent. The kinetic constants in the presence and absence of Mg2+ have been measured and a set of X-ray structures determined at different stages of the reaction. The conformational changes in the active site of the enzyme, induced by Mg2+, substrate binding and substrate carboxylation, have been monitored crystallographically and are discussed. Sulfate ions bound to DTBS may mimic the behaviour of the alpha- and gamma-phosphates of ATP in Mg2+ binding and in the subsequent steps of the reaction. CONCLUSIONS Mg2+ is an essential cation for both substrate binding and carbamate formation by DTBS, when sulfate is present. The conformational changes induced at the active site in the DTBS-substrate complex, when Mg2+ is present, are small yet highly significant and serve to optimize the interactions between substrate and enzyme. DTBS is active as a homodimer and the substrate-binding site straddles both monomers in the dimer. The carboxylation site is unambiguously identified as the N-7 amino group of the substrate, rather than the N-8 amino group, as previously suggested. The elongated nucleotide-binding loop (the P loop) binds both ATP and substrate in a manner which suggests that this feature may be of wider importance.

Rational design of an inhibitor of dethiobiotin synthetase; interaction of 6-hydroxypyrimindin-4(3H)-one with the adenine base binding site

Abstract Modelling of the H-bonding interactions involved in the binding of the adenine base component of ATP to E. coli dethiobiotin synthetase (DTBS) suggested that 6-hydroxypyrimidin-4(3H)-one (6-HP4) should selectively bind to this site in the enzyme. Kinetic studies show that 6-HP4 is a competitive inhibitor of DTBS and x-ray crystallography shows that 6-HP4 binds specifically in the purine base binding site of the enzyme.

Cloning, expression, purification, crystallization and preliminary X-ray characterization of the full- length single-stranded DNA-binding protein from the hyperthermophilic bacterium Aquifex aeolicus

Single-stranded DNA-binding (SSB) proteins stabilize single-stranded DNA, which is exposed by separation of the duplex during DNA replication, recombination and repair. The SSB protein from the hyperthermophile Aquifex aeolicus has been overexpressed in Escherichia coli, purified and characterized and crystals of the full-length protein (147 amino acids; M(r) 17 131.20) have been grown by vapour diffusion from ammonium sulfate pH 7.5 in both the absence and presence of ssDNA [dT(pT)(68)]. All crystals diffract to around 2.9 A resolution and those without bound DNA (native) belong to space group P2(1), with two tetramers in the asymmetric unit and unit-cell parameters a = 80.97, b = 73.40, c = 109.76 A, beta = 95.11 degrees . Crystals containing DNA have unit-cell parameters a = 108.65, b = 108.51, c = 113.24 A and could belong to three closely related space groups (I222, I2(1)2(1)2(1) or I4(1)) with one tetramer in the asymmetric unit. Electrospray mass spectrometry of the crystals confirmed that the protein was intact. Molecular replacement with a truncated E. coli SSB structure has revealed the position of the molecules in the unit cell and refinement of both native and DNA-bound forms is under way.

New mechanism for metal capture by a bacterial protein

Acknowledgements We thank The Wellcome Trust (Edinburgh Protein Interaction Centre), Darwin Trust (Fellowship for DA), University of Edinburgh and CVCP (scholarship, small project grant for travel to ICBIC11, and ORS Award for HZ) for their support for this work. Aims • Most of the Fe3+ in the environment is present as oxyhydroxide clusters and minerals but no molecular mechanism for the capture of such polymers by bacteria has yet been described. • Here we investigate whether Fbp can capture oxyhydroxide Fe3+ complexes.

Mechanistic Studies Of 8-Amino-7-Oxononanoate Synthase.

8-amino-7-oxononanoate synthase is a pyridoxal 5’-phosphate-dependent enzyme which catalyses the decarboxylative condensation of L-alanine with pimeloyl-CoA to form 8-amino-7-oxononanoate. Individual steps in the reaction mechanism ofEscherichia coliAONS have been probed by spectroscopy, pre-steady state kinetics and crystallography. Our results suggest that conformational transitions, induced by substrate and product binding, play an important role in catalysis.