Pedro Matias Lab
Macromolecular Crystallography Unit
Many proteins in nature have either industrial and/or medicinal applications. Knowledge of their three-dimensional structure is essential to understanding their function at the atomic level, and can be used to control or improve their functional activity by the production of small molecules to act as substrates or ligands with specific purposes (e.g., drugs to fight disease) or by engineering selected mutants with enhanced biological activity. Our research program is dedicated to doing just that: determining the 3D structure of selected proteins, and using that knowledge, in combination with other studies (biochemical, spectroscopic, etc.) to understand how these molecules work.
RuvBL1 (RuvB-like 1) and its homolog RuvBL2 are evolutionarily highly conserved AAA+ ATPases essential for many cellular activities. RuvBL1 and RuvBL2 are over expressed in some types of cancer and interact with major oncogenic factors, such as β-catenin and c-Myc, regulating their function. We solved the first crystal structure of the human RuvBL complex with a truncated domain II. The structure is dodecameric, formed by heterohexameric rings with alternating RuvBL1 and RuvBL2 monomers bound to ADP/ATP. Biochemical assays showed that truncation of domain II led to a substantial increase in the ATPase and DNA helicase activities of RuvBL1, RuvBL2 and their complex, strongly suggesting that in vivo activities of these highly interesting therapeutic drug targets are regulated by cofactors inducing conformational changes via domain II in order to modulate the enzyme complex into its active state. This work was carried out in collaboration with the pharmaceutical company Bayer Schering Pharma.
The three-dimensional structure of [NiFeSe] hydrogenase from D. vulgaris Hildenborough in its oxidised, “as isolated” state provided a structural basis for the oxygen tolerance of [NiFeSe] hydrogenases: in the oxidised state there is an exogenous sulphur atom present in the active site, which is bound to the Ni atom and to the Se atom of the selenocysteine. This moves the selenocysteine side-chain conformation into a rotamer that shields the Ni atom from attack by O2 molecules. When the enzyme is activated in production and consumption modes, this sulphur atom can be released as either H2S or HS- and becomes located in a binding site near the active site, which in our structure was found occupied by a Cl- ion. When the enzyme becomes quiescent, the sulphur species can become re-attached to the active site.
NADP+ dependent isocitrate dehydrogenase (IDH; EC 220.127.116.11) belongs to a large family of α-hydroxyacid oxidative β-decarboxylases that catalyze similar two-step reactions, with dehydrogenation to an oxaloacid intermediate preceding β-decarboxylation to an α-ketone product. We obtained the first “fully closed” crystal structures of a pseudo-Michaelis complex of wild-type Escherichia coli IDH (EcoIDH) and the “fully closed” reaction product complex of the K100M mutant, affording a comprehensive view of the induced fit needed for catalysis by comparison with previously obtained “quasi-closed” and “open” conformations. As predicted by others, Lys230* from the second monomer in the biological and crystallographic IDH dimer is positioned to deprotonate/reprotonate the α-hydroxyl in both reaction steps and Tyr160 moves into position to protonate C3 following β-decarboxylation. A proton relay from the catalytic triad Tyr140-Asp307-Lys230* connects the α-hydroxyl of isocitrate to the bulk solvent to complete the picture of the catalytic mechanism.
Mannosyl-3-phosphoglycerate phosphatase (MpgP) from the hyperthermophilic, halotolerant bacterium Thermus thermophilus HB27 is a metal-dependent (Mg2+) Haloalkanoid Acid Dehalogenase-like phosphatase and catalyzes the hydrolysis of mannosyl-3-phosphoglycerate (MPG) into the final product, α-mannosylglycerate. We determined the crystal structure of MpgP in its apo-form and in complex with substrates, substrate analogues and inhibitors. The structural analysis revealed two catalytically relevant enzyme conformations: open (apo-MpgP) and closed (holo-MpgP, in complex with the reaction products), and led to the proposal that in Thermus thermophilus HB27 MpgP the phosphoryl-transfer undergoes a concerted DNSN mechanism with assistance of proton transfer from the general acid Asp8, forming a short-lived PO3- intermediate which is attacked by a nucleophilic water molecule.
- Tiago Bandeiras, iBET Researcher and Project Leader
Sara Silva, Ph.D. student (co-supervision with Tiago Bandeiras)
Sónia Zacarias, Ph.D. student (co-supervision with Inês A. C. Pereira)
Paulo Santo, Ph.D. student (co-supervision with Tiago Bandeiras)
N. Zaarur, X. Xu, P. Lestienne, A.B. Meriin, M. McComb, C.E. Costello, G.P. Newnam, R. Ganti, N.V. Romanova, M. Shanmugasundaram, S.T. Silva, T.M. Bandeiras, P.M. Matias, K.S. Lobachev, I.K. Lednev, Y.O. Chernoff, M.Y. Sherman “RuvbL1 and RuvbL2 enhance aggresome formation and disaggregate amyloid fibrils” (2015) EMBO Journal 112(37):11455-11460.
P.M. Matias, S.H. Baek, T.M. Bandeiras, A. Dutta, W.A. Houry, O. Llorca, J. Rosenbaum “The AAA+ proteins Pontin and Reptin enter adult age: From understanding their basic biology to the identification of selective inhibitors” (2015) Frontiers in Molecular Biosciences 2:17.
S. P. Miller, S. Gonçalves, P. M. Matias, and A. M. Dean, "Evolution of a Transition State: Role of Lys100 in the Active Site of Isocitrate Dehydrogenase" (2014) ChemBioChem, 15:1145-1153
M. C. Marques, R. Coelho, I. A. C. Pereira, P. M. Matias, "Redox state-dependent changes in the crystal structure of [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough" (2013) Int. J. Hydrogen Energ. 38(21):8664–8682. http://dx.doi.org/j.ijhydene.2013.04.132
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Muitas proteínas na Natureza podem ter aplicações medicinais e industriais. O conhecimento da sua estrutura tridimensional é essencial para a compreensão da sua função biológica ao nível atómico, e este conhecimento pode igualmente ser utilizado para controlar ou melhorar a sua função, quer através da produção de moléculas pequenas que funcionam como substratos ou ligandos com fins específicos (p.ex., fármacos para combater doenças), quer através da produção de mutantes seleccionados com maior actividade biológica. O programa de investigação deste Laboratório assenta precisamente nessas duas vertentes: na determinação da estrutura tridimensional de proteínas seleccionadas, e na utilização desse conhecimento, em combinação com outros estudos envolvendo p.ex. métodos bioquímicos e espectroscópicos, para a compreensão do funcionamento dessas moléculas. Estes estudos envolvem a colaboração com outros Laboratórios e são executados no âmbito de projectos académicos ou de parcerias com empresas farmacêuticas.