Christopher M. Bruns

Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase

Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca2+/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg1400, and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.

Structure of Haemophilus influenzae Fe(+3)-binding protein reveals convergent evolution within a superfamily.

The first crystal structure of the iron-transporter ferric ion-binding protein from Haemophilus influenzae (hFBP), at 1.6 Å resolution, reveals the structural basis for iron uptake and transport required by several important bacterial pathogens. Paradoxically, although hFBP belongs to a protein superfamily which includes human transferrin, iron binding in hFBP and transferrin appears to have developed independently by convergent evolution. Structural comparison of hFBP with other prokaryotic periplasmic transport proteins and the eukaryotic transferrins suggests that these proteins are related by divergent evolution from an anion-binding common ancestor, not from an iron-binding ancestor. The iron binding site of hFBP incorporates a water and an exogenous phosphate ion as iron ligands and exhibits nearly ideal octahedral metal coordination. FBP is highly conserved, required for virulence, and is a nodal point for free iron uptake in several Gram-negative pathogenic bacteria, thus providing a potential target for broad-spectrum antibacterial drug design against human pathogens such as H. influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis.

Characterization of the electrophile binding site and substrate binding mode of the 26-kDa glutathione S-transferase from Schistosoma japonicum

The 26‐kDa glutathione S‐transferase from Schistosoma japonicum (Sj26GST), a helminth worm that causes schistosomiasis, catalyzes the conjugation of glutathione with toxic secondary products of membrane lipid peroxidation. Crystal structures of Sj26GST in complex with glutathione sulfonate (Sj26GSTSLF), S‐hexyl glutathione (Sj26GSTHEX), and S‐2‐iodobenzyl glutathione (Sj26GSTIBZ) allow characterization of the electrophile binding site (H site) of Sj26GST. The S‐hexyl and S‐2‐iodobenzyl moieties of these product analogs bind in a pocket defined by side‐chains from the β1‐α1 loop (Tyr7, Trp8, Ile10, Gly12, Leu13), helix α4 (Arg103, Tyr104, Ser107, Tyr111), and the C‐terminal coil (Gln204, Gly205, Trp206, Gln207). Changes in the Ser107 and Gln204 dihedral angles make the H site more hydrophobic in the Sj26GSTHEX complex relative to the ligand‐free structure. These structures, together with docking studies, indicate a possible binding mode of Sj26GST to its physiologic substrates 4‐hydroxynon‐2‐enal (4HNE), trans‐non‐2‐enal (NE), and ethacrynic acid (EA). In this binding mode, hydrogen bonds of Tyr111 and Gln207 to the carbonyl oxygen atoms of 4HNE, NE, and EA could orient the substrates and enhance their electrophilicity to promote conjugation with glutathione. Proteins 2003;51:137–146.

Methods for Building and Refining 3D Models of RNA

Interest in RNA has grown tremendously in recent years as we uncover more and more roles for RNA in the cell. Investigation of RNA function is often hampered by the absence of even a tentative 3D structure which can guide experiments. Experimental structure determination is difficult because of RNA’s large size, high charge and flexibility, propensity for kinetic trapping, and the lack of the distinctive surface features necessary for crystallization. Computational structure prediction is challenging for mostly the same reasons. In this work, we describe three methods which are used in different ways to predict the structure and dynamics of RNA. RNABuilder is an “erector set” for constructing RNA molecules based on experiments, hypotheses, or other information known to the user. NAST quickly produces ensembles of coarse-grained molecules based on the statistics of backbone conformation. Lastly, Zephyr uses the graphical processing unit rather than the CPU to speed up conventional molecular dynamics calculations.