Towards high-resolution structural biology of membrane protein complexes in their native lipid environment

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications & trials

Abstract

(1) Structural and functional investigation of CXCR3 chemokine receptor in its role in recurrent miscarriage Recurrent miscarriage (RM) is usually defined as the loss of three or more consecutive pregnancies prior to the 20th week of gestation affecting approximately 1% of women of reproductive age. 50% of cases of RM is unknown but there is evidence supporting immune causes, more specifically that a T helper (Th) 1-type response is associated with the pathogenesis of RM. RM woman show higher ratios of Th1 (CXCR3 and CCR5) to Th2 (CCR3 and CCR4) chemokine receptors. A Th1-type reaction in the maternofetal interface mainly triggers an inflammatory response, while a Th2-type reaction typically promotes growth of trophoblastic cells which is beneficial for successful maintenance of a pregnancy. Incoming postdoc Dr. Munazza Shahid will study the structure and function of CXCR3 chemokine receptor alone and in complex with its ligands (CXCL4, CXCL9, CXCL10 and CXCL11) to work towards a treatment to prevent pregnancy loss in women with RM. (2) Structure and function of magnesium channel Mrs2 Mrs2 is the eukaryotic homolog of bacterial magnesium channel CorA. CorA forms a homo-pentameric channel which forms a symmetric closed state at normal to high concentrations of magnesium with magnesium binding sites between protomers as well as near the membrane pore. Under low magnesium concentrations the channel undergoes an asymmetric opening likely caused by the destabilization of protomer interactions when magnesium ions dissociate from their binding site. Incoming postdoc Dr. Louis Lai will expand research on magnesium channels by looking at eukaryotic magnesium channel Mrs2 which is located in the inner mitochondrial membrane. Structural studies in synthetic as well as native nanodiscs as well as liposomes are planned to investigate the structure and mechanism of this eukaryotic channel. (3) Structural determination of full-length SARS-CoV-2 spike protein and drug development COVID-19 caused by the SARS-CoV-2 virus has posed a global threat since it was first identified end of 2019. The rapid development of vaccines helped to counteract the rapid spread of COVID-19. However, vaccines for children under the age of 12 have not yet been approved. More children have been infected by more contagious variants of the virus and the recent surge of COVID-19 cases has put an unprecedented pressure on the pediatric health care system. The SARS-Cov-2 spike protein is responsible for the initial binding of the virus to the receptor ACE2 on human cells. Better understanding of the function and structure of the spike protein is critical for development of both primary prevention such as vaccine, and therapeutic treatments to combat the COVID-19 pandemic. Structures of the spike proteins soluble ectodomain have been determined but the full-length spike including its membrane domain has not been well studied. We are working towards determining the structures of full-length spike protein and identify the key vaccine and drug binding interfaces in order to develop treatments that block viral entry into human cells with high efficiency and specificity which are also safe for children. We have successfully cloned and expressed the full-length spike protein, and we are working towards high-resolution structural determination of different variants and complexes. (4) Structural investigation of inner mitochondrial membrane supercomplex II and III As the powerhouses of cells, mitochondria provide energy in form of ATP for most of the cellular activities. There are five essential protein complexes located on the inner mitochondrial membrane which carry out one of the most important reaction in the cells oxidative phosphorylation, which generates ATP. Functions of these protein complexes have been extensively studied and crystal structures of individual complexes have been determined. It is proposed that protein complexes of the respiratory chain can associate and form of supercomplexes or respirasomes. Among these five protein complexes, cytochrome bc1, also known as complex III, is a central component of the cellular respiratory chain. It catalyzes electron transfer from quinol to cytochrome c and couples this electron transfer process to proton translocation across the membrane. Mitochondrial Complex II, also called succinate dehydrogenase, is another important protein complex within the mitochondrial electron transfer chain. It oxidizes succinate from the Krebs-cycle to fumarate and reduces ubiquinone to ubiquinol. Previous studies have proposed the formation of a supercomplex between complex II and complex III, which has been confirmed by biochemical analyses of scientist Dr. Fei Zhou. We are currently working towards solving the structure of this supercompex by Single-Particle Cryo-EM. Together with biochemical data, the structure of supercompex II and III will reveal detailed information of electron transfer between the Krebs-cycle and the respiratory chain and could shed light on the biogenesis and treatment of many human diseases related to respiratory chain deficiency such as delayed child development, autism, tumorigenesis and aging. (5) Collaborations Collaborations involving structural and computational studies on a variety of membrane proteins including transporters, channels, and receptors as well as viral spike protein conformations in different cellular compartments, virus-like-particle (VLP), SARS-CoV-2 accessory membrane protein, extracellular vesicles and lipid transport across cells, as well as testing new detergents and polymers to gently extract membrane protein complexes from their native lipid environment for high-resolution structural studies.