James W Coulton
贰尘别谤颈迟耻蝉听笔谤辞蹿别蝉蝉辞谤
Microbial Physiology/ Genetics
RETIRED: No longer accepting graduate students or post-graduated trainees
Appointments
- Co-chair of听CREATE-CDMC
- Member of the听Scientific Committee
Research Orientations
Research Orientation 1
Structural biology of bacterial membrane protein complexes
The Coulton research group studies membrane proteins (MPs) that are required for transport of iron, an essential nutrient, across the bacterial cell envelope.听 Our on-going research collaborations with colleagues world-wide emphasize structural determinants of MPs required for transport, including solving their 3-D structures by X-ray crystallography.
For import of iron-siderophore complexes, seven proteins in the cell envelope of听Escherichia coli听are essential.听 We use FhuA from听E. coli听as a model bacterial outer MP.听 TonB, partner protein of FhuA, is one of three proteins from the energy-transducing complex TonB鈥揈xbB鈥揈xbD that is embedded in the cytoplasmic membrane.听 Initial studies of the TonB interactome began with our X-ray structure 2GRX for the co-crystal of TonB鈥揊huA.听 We recently adopted complementary strategies to generate models for the 3-D organization of the TonB interactome.听 Having isolated abundant (mg) quantities of purified ExbB鈥揈xbD complexes and substituted amphipols (APol) for detergents, we collected low-resolution data: small angle X-ray scattering (SAXS) and small angle neutron scattering (SANS) (Sverzhinsky听et al., Journal of Membrane Biology 2014).听 Medium resolution electron microscopy resolved particles (10 nm diameter) by negative staining (Sverzhinsky听et al., Structure 2014; Journal of Bacteriology 2015).听 Remarkably, the stoichiometry of ExbB4鈥揈xbD2 changed on addition of TonB: ExbD4鈥揈xbD1鈥揟onB1.听 The accompanying video听 shows a scale model for interaction of known proteins of听E. coli听that participate in transport of iron-siderophores across the cell envelope.听 Video credit: A. Sverzhinsky.
Our steepest challenge is to grow 3-D crystals, a partnership with I. Moraes at the Membrane Protein Laboratory,听Diamond Light Source, Oxford; S. Iwata, Director.听 Growing MP crystals requires novel strategies and leading infrastructure: lipidic cubic phase and screening hundreds of conditions with nano-robotics.听 Crystals must be of sufficient quality that they diffract; to date we observe diffraction to 2.9 angstroms at the microfocus beamline I24, Diamond Light Source.听 Unlike crystals that are grown for soluble proteins (usually to 100 碌m in size), the MP crystals of the ExbB鈥揈xbD complex that we reproducibly grow in lipidic cubic phase are 鈥渟howers鈥�, only 5 to 10 碌m in all dimensions.听 Our 2015 visits to world-unique beam lines,听X-ray free electron laser XFEL,听at Stanford University, and at听SPring-8/SACLA in Japan听provided opportunities for data collection on the ExbB-ExbD complex.
When we fully understand the structure and function of TonB, ExbB, and ExbD, then we will know a critical mechanism whereby Gram-negative bacteria acquire iron.听 Knowledge advanced by outcomes from our research will enable the design of antibacterial compounds that block iron import, thus markedly slowing bacterial growth.
Ref.:听 A. Sverzhinsky, Ph.D. thesis 2015, 麻豆AV
Protein鈥損rotein interactions for early intracellular vitamin B12 metabolism in mammals
Vitamin B12, or cobalamin, is a water-soluble vitamin required as cofactor for two mammalian enzymatic processes: methionine regeneration in the cytoplasm by methionine synthase (MS), and fatty acid/amino acid metabolism in mitochondria by methylmalonyl-CoA mutase (MCM).听 While the molecular nature of intracellular cobalamin metabolism in mammals remains poorly understood, the proteins MMACHC, MMADHC, LMBD1 and ABCD4 are implicated in its early uptake and processing.听 Due to the inherent challenges associated with the cellular utilization of this cofactor, we propose that these proteins mediate its early intracellular channeling; the objective of this thesis was to characterize the protein-protein interactions that coordinate this process.
To gain insight into the function of MMADHC, recombinant isoforms were purified and low-resolution structural features were determined.听 MMADHC is monomeric and adopts an extended conformation in solution, with regions of disorder identified at the N-terminal domain.听 Panning combinatorial phage libraries against recombinant MMADHC allowed mapping of putative sites of interaction on each protein.听 Kinetic analyses using surface plasmon resonance (SPR) confirmed a sub-micromolar affinity for the MMACHC鈥揗MADHC interaction.听 Based on these studies, we propose (Deme听et al.听Molecular Genetics and Metabolism 2012) that the function of MMADHC is exerted through its structured C-terminal domain听via听interactions with MMACHC in the cytoplasm.
Clinical phenotypes and subcellular location of MS and MUT dictate that MMACHC functions in the cytoplasm while MMADHC functions at a branch point in the pathway in both the cytoplasm and the mitochondrion.听 To demonstrate that the MMACHC鈥揗MADHC interaction is physiologically plausible, we used subcellular fractionation and immunofluorescence to confirm that MMACHC is cytoplasmic while MMADHC is dual-localized to the cytoplasm and to mitochondria (Mah听et al.听Molecular Genetics and Metabolism 2013).
Protein interaction analyses were extended by our recombinant production of the lysosomal membrane proteins LMBD1 and ABCD4.听 Detergent-solubilized LMBD1 and ABCD4 formed homodimers in solution.听 SPR provided direct听in vitro听binding data for an LMBD1鈥揂BCD4 interaction with low nanomolar affinity.听 Consistent with our phage display predictions, MMACHC interacted with LMBD1 and with ABCD4 at high affinity (Deme听et al.听 Molecular Membrane Biology 2014).
Our results support a model whereby membrane-bound LMBD1 and ABCD4 regulate vectorial delivery of lysosomal cobalamin to cytoplasmic MMACHC, preventing cofactor dilution to the cytosolic milieu and protecting against inactivating side reactions.听 Subsequent formation of a cytoplasmic MMACHC鈥揗MADHC complex then processes and partitions this cofactor to the downstream enzymes MCM and MS.听 These studies identify and characterize multiprotein complexes, advancing our basic understanding of early intracellular cobalamin metabolism.
Ref.: J.C. Deme, Ph.D. thesis 2014, 麻豆AV
- Sverzhinsky A, Chung JW, Deme JC, Fabre L, Levey KT, Plesa M, Carter DM, Lypaczewski P,听Coulton JW. 听(2015)听.听Journal of Bacteriology听197:1873-1885.
- Sverzhinsky A, Qian S, Yang L, Allaire M, Moraes I, Ma D, Chung JW, Zoonens M, Popot J-L,听Coulton JW. (2014)听Journal of Membrane Biology听247:1005-1018.
- Sverzhinsky A, Fabre L, Cottreau AL, Biot-Pelletier DM, Khalil S, Bostina M, Rouiller I,听Coulton JW. (2014)听听.听听Structure听22:791-797.
- Deme JC, Hancock MA, Xia X, Sintre CA, Plesa M, Kim JC, Carpenter EP, Rosenblatt DS,听Coulton JW.听(2014)听Molecular Membrane Biology听31:250-261.
- Mills A, Le HT,听Coulton JW, Duong F. (2014)听听听Biochimica et Biophysica Acta.听1838(1 Pt B):364-371.
- Mah W, Deme JC, Watkins D, Fung S, Janer A, Shoubridge EA, Rosenblatt DS,听Coulton JW.听(2013)听听Molecular Genetics and Metabolism听108:112-118.
- Deme JC, Miousse IR, Plesa M, Kim JC, Hancock MA, Mah W, Rosenblatt DS,听Coulton JW.听(2012)听听听Molecular Genetics and Metabolism听107:352-362.
Career listing on PubMed:听.