Biophysical studies including X-ray structure determination of the Mitochondrial Pyruvate Carrier (MPC) complex

• Grant number: 14/06954-9
• Support Opportunities: Scholarships in Brazil - Post-Doctorate
• Effective date (Start): December 01, 2014
• Effective date (End): January 09, 2018
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Andre Luis Berteli Ambrosio
• Grantee: Raghavendra Sashi Krishna Nagampalli
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovações (Brasil). Campinas , SP, Brazil
• Associated scholarship(s): 17/02391-8 - Three-dimensional structure determination of human MPC2 by X-ray crystallography - a mechanistic view into mitochondrial pyruvate transport, BE.EP.PD

Abstract

Pyruvate is the end product of cytosolic glycolysis and has a number of possible intracellular fates, the major one being mitochondrial transport. Pyruvate transport across the mitochondrial membrane is a critical step in carbohydrate, amino acid, and lipid metabolism. While pyruvate has been known to cross the mitochondrial membrane over a long period of time, it is only recently that proteins necessary for this activity have been identified. In this perspective, two internal mitochondrial membrane (IMM) proteins, named MPC1 and MPC2, were identified as the proteins essential for the transport of pyruvate in yeast (S. cerevisiae), Drosophila and humans. These proteins were believed to possess three transmembrane helices with each subunits weighing 15 kDa. However, according to the data reported, the functional complex (MPC1 and MPC2) is believed to have a molecular weight of 150 kDa indicating the possibility of a decamer formation. Although the proteins involved in the pyruvate transport have been identified, questions such as what are the role of the individual proteins (MPC 1 and MPC 2) in the complex, what is the stoichiometry between them, and what are the molecular determinants of the pyruvate transport have not been addressed so far. In order to answer these questions, the present project proposes the biophysical characterization along with, within the available time, the structure determination of the MPC complex. Considering that MPC is a transmembrane complex, one would expect serious difficulties in the expression, purification and structural studies by X-ray crystallography. To overcome these difficulties, yeast expression will be employed as the choice of the expression system as it could help with the folding and stability of the MPC complex. In this regard, during the year of 2013 our laboratory generated a set of preliminary results that support In addition to this, determination of the detergent or mixture of detergents which will effectively break the organization of the plasma membrane, without compromising the tertiary structure of the protein, also need be determined. Biophysical and biochemical studies (Analytical ultra-centrifugation, isothermal titration calorimety, surface plasmonic resonance, and negative staining and/or cryo-electron microscopy) will be carried out on the purified MPC complex, in order to determine its exact molecular weight and stoichiometry and overall architecture. As is often in the case of protein crystallization and structure determination, the choice for the right construct is a key between the success and failure. Hence the synthetic genes of the complex (MPC1 and MPC2) from ten different species were already purchased. Crystallization trials will be carried out on the most promising constructs, followed by the unprecedented crystal structure determination of MPC complex. The proposed project once successfully completed has a potential to provide valuable insights in understanding underlying mechanisms of pyruvate shuttling across the mitochondrial membrane.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

KRISHNA NAGAMPALLI, RAGHAVENDRA SASHI; NECIOSUP QUESNAY, JOSE EDWIN; ADAMOSKI, DOUGLAS; ISLAM, ZEYAUL; BIRCH, JAMES; SEBINELLI, HEITOR GOBBI; BRUNO MOREIRA GIRARD, RICHARD MARCEL; RODRIGUES ASCENCAO, CAROLLINE FERNANDA; FALA, ANGELA MARIA; PAULETTI, BIANCA ALVES; et al. Human mitochondrial pyruvate carrier 2 as an autonomous membrane transporter. SCIENTIFIC REPORTS, v. 8, FEB 22 2018. (17/02391-8, 14/20673-2, 16/06034-2, 15/02734-7, 15/25832-4, 14/17820-3, 14/12663-7, 14/06954-9)

QUESNAY, JOSE EDWIN NECIOSUP; POLLOCK, NAOMI L.; NAGAMPALLI, RAGHAVENDRA SASHI KRISHNA; LEE, SARAH C.; BALAKRISHNAN, VIJAYAKUMAR; DIAS, SANDRA MARTHA GOMES; MORAES, ISABEL; DAFFORN, TIM R.; AMBROSIO, ANDRE LUIS BERTELI. Insights on the Quest for the Structure-Function Relationship of the Mitochondrial Pyruvate Carrier. BIOLOGY-BASEL, v. 9, n. 11, NOV 2020. (14/20673-2, 17/11766-5, 19/02261-2, 13/07600-3, 14/06954-9, 18/00492-4, 17/02391-8, 15/02734-7)