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Instruments / Technical Area	Publication Details	Published
B21-High Throughput SAXS	Solution and membrane interaction dynamics of Mycobacterium tuberculosis fatty Acyl-CoA synthetase FadD13 Camilla A. K. Lundgren , Michael Lerche , Charlotta Norling , Martin Högbom Biochemistry DOI: 10.1021/acs.biochem.0c00987 Diamond Proposal Number(s): [11265] Open Access Show Abstract ▼ Abstract: The very-long-chain fatty acyl-CoA synthetase FadD13 from Mycobacterium tuberculosis activates fatty acids for further use in mycobacterial lipid metabolism. FadD13 is a peripheral membrane protein, with both soluble and membrane-bound populations in vivo. The protein displays a distinct positively charged surface patch, suggested to be involved in membrane association. In this paper, we combine structural analysis with liposome co-flotation assays and membrane association modeling to gain a more comprehensive understanding of the mechanisms behind membrane association. We show that FadD13 has affinity for negatively charged lipids, such as cardiolipin. Addition of a fatty acid substrate to the liposomes increases the apparent affinity of FadD13, consistent with our previous hypothesis that FadD13 can utilize the membrane to harbor its very-long-chain fatty acyl substrates. In addition, we unambiguously show that FadD13 adopts a dimeric arrangement in solution. The dimer interface partly buries the positive surface patch, seemingly inconsistent with membrane binding. Notably, when cross-linking the dimer, it lost its ability to bind and co-migrate with liposomes. To better understand the dynamics of association, we utilized two mutant variants of FadD13, one in which the positively charged patch was altered to become more negative and one more hydrophobic. Both variants were predominantly monomeric in solution. The hydrophobic variant maintained the ability to bind to the membrane, whereas the negative variant did not. Taken together, our data indicate that FadD13 exists in a dynamic equilibrium between the dimer and monomer, where the monomeric state can adhere to the membrane via the positively charged surface patch.	Apr 2021
B21-High Throughput SAXS I24-Microfocus Macromolecular Crystallography	Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens Vivek Srinivas , Hugo Lebrette , Daniel Lundin , Yuri Kutin , Margareta Sahlin , Michael Lerche , Jürgen Eirich , Rui M. M. Branca , Nicholas Cox , Britt-Marie Sjöberg , Martin Högbom Nature 47 DOI: 10.1038/s41586-018-0653-6 Diamond Proposal Number(s): [11265, 15806] Show Abstract ▼ Abstract: Ribonucleotide reductase (RNR) catalyses the only known de novo pathway for the production of all four deoxyribonucleotides that are required for DNA synthesis1,2. It is essential for all organisms that use DNA as their genetic material and is a current drug target3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a dinuclear metal site has been viewed as necessary to generate and stabilize the catalytic radical that is essential for RNR activity5,6,7. Here we describe a group of RNR proteins in Mollicutes—including Mycoplasma pathogens—that possess a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable 3,4-dihydroxyphenylalanine (DOPA) radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase. The metal-independent radical requires new mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. It is possible that this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms that encode this type of RNR—some of which are developing resistance to antibiotics—are involved in diseases of the respiratory, urinary and genital tracts. Further characterization of this RNR family and its mechanism of cofactor generation will provide insight into new enzymatic chemistry and be of value in devising strategies to combat the pathogens that utilize it. We propose that this RNR subclass is denoted class Ie.	Oct 2018
I02-Macromolecular Crystallography I03-Macromolecular Crystallography	Structure and cooperativity of the cytosolic domain of the CorA Mg2+ channel from Escherichia coli Michael Lerche , Hena Sandhu , Lukas Flöckner , Martin Högbom , Mikaela Rapp Structure DOI: 10.1016/j.str.2017.05.024 Diamond Proposal Number(s): [15806] Show Abstract ▼ Abstract: Structures of the Mg2+ bound (closed) and apo (open) states of CorA suggests that channel gating is accomplished by rigid-body motions between symmetric and asymmetric assemblies of the cytosolic portions of the five subunits in response to ligand (Mg2+) binding/unbinding at interfacial sites. Here, we structurally and biochemically characterize the isolated cytosolic domain from Escherichia coli CorA. The data reveal an Mg2+-ligand binding site located in a novel position between each of the five subunits and two Mg2+ ions trapped inside the pore. Soaking experiments show that cobalt hexammine outcompetes Mg2+ at the pore site closest to the membrane. This represents the first structural information of how an analog of hexa-hydrated Mg2+ (and competitive inhibitor of CorA) associates to the CorA pore. Biochemical data on the isolated cytoplasmic domain and full-length protein suggests that gating of the CorA channel is governed cooperatively.	Jan 2017

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