Klaus Fütterer

The C-terminal domain of full-length E. coli SSB is disordered even when bound to DNA.

The crystal structure of full‐length homotetrameric single‐stranded DNA (ssDNA)‐binding protein from Escherichia coli (SSB) has been determined to 3.3 Å resolution and reveals that the entire C‐terminal domain is disordered even in the presence of ssDNA. To our knowledge, this is the first experimental evidence that the C‐terminal domain of SSB may be inherently disordered. The N‐terminal DNA‐binding domain of the protein is well ordered and is virtually indistinguishable from the previously determined structure of the chymotryptic fragment of SSB (SSBc) in complex with ssDNA. The absence of observable interactions with the core protein and the crystal packing of SSB together suggest that the disordered C‐terminal domains likely extend laterally away from the DNA‐ binding domains, which may facilitate interactions with components of the replication machinery in vivo. The structure also reveals the conservation of molecular contacts between successive tetramers mediated by the L45 loops as seen in two other crystal forms of SSBc, suggesting a possible functional relevance of this interaction.

Structure of N-myristoyltransferase with bound myristoylCoA and peptide substrate analogs.

N-myristoyltransferase (Nmt) attaches myristate to the N-terminal glycine of many important eukaryotic and viral proteins. It is a target for anti-fungal and anti-viral therapy. We have determined the structure, to 2.9 Å resolution, of a ternary complex of Saccharomyces cerevisiae Nmt1p with bound myristoylCoA and peptide substrate analogs. The model reveals structural features that define the enzymes substrate specificities and regulate the ordered binding and release of substrates and products. A novel catalytic mechanism is proposed involving deprotonation of the N-terminal ammonium of a peptide substrate by the enzymes C-terminal backbone carboxylate.

The structure of myristoyl-CoA : protein N-myristoyltransferase

Protein N-myristoylation is a covalent modification that occurs co-translationally in eukaryotes. Myristate, a rare 14 carbon saturated fatty acid (C14:0), is attached, via an amide linkage, to the N-terminal glycine of a subset of eukaryotic and viral proteins by myristoyl-CoA:protein N-myristoyltransferase (Nmt). Genetic and biochemical studies have established that Nmt is a target for development of a new class of fungicidal drugs. The enzyme is also a potential target for development of antiviral and antineoplastic agents. The structure of Saccharomyces cerevisiae Nmt1p has been determined recently with bound substrate analogs. The Nmt fold resembles the fold of members of the GCN5-related N-acetyltransferase superfamily. The structure reveals how Nmts myristoyl-CoA and peptide substrates are recognized and bound, and what elements control the enzymes ordered kinetic mechanism. Acyl transfer occurs through the nucleophilic addition-elimination reaction: an oxyanion hole formed by main chain atoms polarizes the thioester carbonyl and stabilizes the transition state while deprotonation of the ammonium of the Gly acceptor appears to be mediated by Nmts C-terminal carboxylate. The use of main chain carboxylate atoms as general base catalyst is a novel feature.

9 Biology and enzymology of protein N-myristoylation

Publisher Summary This chapter reviews a number of studies that have advanced the understanding of how proteins are N-myristoylated and how N-myristoyl proteins function. Thermodynamic analyses of the interactions of acyl peptides and acyl proteins with model membranes have confirmed the importance of the limited hydrophobicity of myristate in regulating dynamic interactions between N-myristoyl proteins and their membrane or protein partners. Genetic studies have shown that protein N-myristoylation is essential for the survival of Saccharomyces cerevisiae during periods when nutrients are unlimited, as well as during periods when nutrients are scarce. Genetic studies have also shown that protein N-myristoylation is required for the survival of the common human fungal pathogens Candida albicans and Cryptococcus neoformans . Analyses of the enzyme responsible for catalyzing protein N-myristoylation—myristoyl-CoA, protein N-myristoyltransferase (Nmt)—have disclosed that orthologous Nmts have subtle differences in their peptide substrate specificities and that these differences can be exploited to generate species-selective inhibitors of fungal Nmts that are fungicidal. X-Ray crystallographic determination of the structure of Nmt has revealed how this enzyme interacts with its substrates, and how catalysis occurs through a novel mechanism.

Crystallographic phasing of myristoyl-CoA-protein N-myristoyltransferase using an iodinated analog of myristoyl-CoA

Myristoyl-CoA-protein N-myristoyltransferase (Nmt; E.C. 2.1.3.97) catalyzes the covalent attachment of myristate to the N-terminal glycine amine of many eukaryotic and viral proteins. The molecular structure of the ternary complex of Saccharomyces cerevisiae Nmt1p with a bound non-hydrolyzable myristoyl-CoA analog, S-(2-oxopentadecyl)-CoA, and a competitive peptidomimetic inhibitor, SC-58272, was solved to 2.9 A resolution by X-ray crystallography. The structure determination utilized diffraction data from an iodinated ternary complex in which a newly designed and synthesized compound, S-(13-iodo-2-oxotridecyl)-CoA, was substituted for S-(2-oxopentadecyl)-CoA. Replacing the two terminal fatty acid C atoms of myristate by iodine produced, under the same crystallization conditions, heavy-atom-derivatized crystals of defined site occupancy that were isomorphous to the native complex. This approach for obtaining experimental phase information can be extended to other crystal structures of protein-fatty acyl complexes. The synthesis of S-(13-iodo-2-oxotridecyl)-CoA and the phasing procedure are described.