ABSTRACT
Acetyl-CoA carboxylase (ACC) catalyses the first committed step in fatty acid biosynthesis: a metabolic pathway required for several important biological processes including the synthesis and maintenance of cellular membranes. ACC employs a covalently attached biotin moiety to bind a carboxyl anion and then transfer it to acetyl-CoA, yielding malonyl-CoA. These activities occur at two different subsites: the biotin carboxylase (BC) and carboxyltransferase (CT). Structural biology, together with small molecule inhibitor studies, has provided new insights into the molecular mechanisms that govern ACC catalysis, specifically the BC and CT subunits. Here, we review these recent findings and highlight key differences between the bacterial and eukaryotic isozymes with a view to establish those features that provide an opportunity for selective inhibition. Especially important are examples of highly selective small molecule inhibitors capable of differentiating between ACCs from different phyla. The implications for early stage antibiotic discovery projects, stemming from these studies, are discussed.
Subject(s)
Acetyl-CoA Carboxylase , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Bacteria/enzymology , Enzyme Inhibitors/pharmacology , Acetyl-CoA Carboxylase/antagonists & inhibitors , Acetyl-CoA Carboxylase/chemistry , Acetyl-CoA Carboxylase/genetics , Acetyl-CoA Carboxylase/metabolism , Anti-Bacterial Agents/chemistry , Enzyme Inhibitors/chemistry , Fatty Acids/biosynthesis , Humans , Models, MolecularABSTRACT
In Bacillus subtilis, the termination of DNA replication via polar fork arrest is effected by a specific protein:DNA complex formed between the replication terminator protein (RTP) and DNA terminator sites. We report the crystal structure of a replication terminator protein homologue (RTP.C110S) of B. subtilis in complex with the high affinity component of one of its cognate DNA termination sites, known as the TerI B-site, refined at 2.5 A resolution. The 21 bp RTP:DNA complex displays marked structural asymmetry in both the homodimeric protein and the DNA. This is in contrast to the previously reported complex formed with a symmetrical TerI B-site homologue. The induced asymmetry is consistent with the complex's solution properties as determined using NMR spectroscopy. Concomitant with this asymmetry is variation in the protein:DNA binding pattern for each of the subunits of the RTP homodimer. It is proposed that the asymmetric "wing" positions, as well as other asymmetrical features of the RTP:DNA complex, are critical for the cooperative binding that underlies the mechanism of polar fork arrest at the complete terminator site.
Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/chemistry , DNA, Bacterial , DNA-Binding Proteins/chemistry , Nucleic Acid Conformation , Protein Structure, Tertiary , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Crystallography, X-Ray , DNA Replication , DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Models, Molecular , Molecular Sequence DataABSTRACT
The replication terminator protein (RTP) of Bacillus subtilis binds to specific DNA sequences that halt the progression of the replisome in a polar manner. These terminator complexes flank a defined region of the chromosome into which they allow replication forks to enter but not exit. Forcing the fusion of replication forks in a specific zone is thought to allow the coordination of post-replicative processes. The functional terminator complex comprises two homodimers each of 29 kDa bound to overlapping binding sites. A preparation of RTP and a 37-base-pair TerI sequence (comprising two binding sites for RTP) has been purified and crystallized. A data set to 3.9 A resolution with 97.0% completeness and an R(sym) of 12% was collected from a single flash-cooled crystal using synchrotron radiation. The diffraction data are consistent with space group P622, with unit-cell parameters a = b = 118.8, c = 142.6 A.
Subject(s)
Bacillus subtilis/chemistry , Bacterial Proteins/chemistry , DNA, Bacterial/chemistry , DNA-Binding Proteins/chemistry , Bacillus subtilis/genetics , Bacterial Proteins/isolation & purification , Base Pairing , Binding Sites , Crystallization , DNA, Bacterial/genetics , DNA-Binding Proteins/isolation & purification , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Terminator Regions, Genetic , X-Ray DiffractionABSTRACT
The alphaCP family of proteins [also known as poly(C)-binding or heterogeneous nuclear ribonucleoprotein E proteins] are involved in the regulation of messenger RNA (mRNA) stability and translational efficiency. They bind via their triple heterologous nuclear ribonucleoprotein K homology (KH) domain structures to C-rich mRNA, and are thought to interact with other mRNA-binding proteins as well as provide direct nuclease protection. In particular, alphaCP1 and alphaCP2 have been shown to bind to a specific region of androgen receptor (AR) mRNA, resulting in its increased stability. The roles of each of the KH motifs in the binding affinity and the specificity is not yet understood. We report the beginning of a systematic study of each of the alphaCP KH domains, with the cloning and expression of alphaCP1-KH2 and alphaCP1-KH3. We report the ability of alphaCP1-KH3, but not alphaCP1-KH2, to bind the target AR mRNA sequence using an RNA electrophoretic mobility gel shift assay. We also report the preparation of an alphaCP1-KH3/AR mRNA complex for structural studies. (1)H-(15)N heteronuclear single quantum correlation NMR spectra of (15)N-labelled alphaCP1-KH3 verified the integrity and good solution behaviour of the purified domain. The titration of the 11-nucleotide RNA target sequence from AR mRNA resulted in a rearrangement of the (1)H-(15)N correlations, demonstrating the complete binding of the protein to form a homogeneous protein/RNA complex suitable for future structural studies.
Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein K/chemistry , RNA/chemistry , Amino Acid Motifs , Binding Sites , Biophysics/methods , Cell Line, Tumor , DNA-Binding Proteins , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Glutathione Transferase/metabolism , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/chemistry , Humans , Kinetics , Macromolecular Substances/chemistry , Magnetic Resonance Spectroscopy , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , RNA/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins , Receptors, Androgen/metabolism , Temperature , Time FactorsABSTRACT
Poly(C)-binding proteins (CPs) are important regulators of mRNA stability and translational regulation. They recognize C-rich RNA through their triple KH (hn RNP K homology) domain structures and are thought to carry out their function though direct protection of mRNA sites as well as through interactions with other RNA-binding proteins. We report the crystallographically derived structure of the third domain of alphaCP1 to 2.1 A resolution. alphaCP1-KH3 assumes a classical type I KH domain fold with a triple-stranded beta-sheet held against a three-helix cluster in a betaalphaalphabetabetaalpha configuration. Its binding affinity to an RNA sequence from the 3'-untranslated region (3'-UTR) of androgen receptor mRNA was determined using surface plasmon resonance, giving a K(d) of 4.37 microM, which is indicative of intermediate binding. A model of alphaCP1-KH3 with poly(C)-RNA was generated by homology to a recently reported RNA-bound KH domain structure and suggests the molecular basis for oligonucleotide binding and poly(C)-RNA specificity.
Subject(s)
3' Untranslated Regions/chemistry , Models, Molecular , RNA-Binding Proteins/chemistry , 3' Untranslated Regions/metabolism , Amino Acid Sequence , Binding Sites , Molecular Sequence Data , Oligonucleotides/chemistry , Poly C/chemistry , Protein Structure, Tertiary , RNA-Binding Proteins/metabolism , Sequence Alignment , Surface Plasmon ResonanceABSTRACT
We report the structural and biophysical consequences of cysteine substitutions in the DNA-binding replication terminator protein (RTP) of Bacillus subtilis, that resulted in an optimised RTP mutant suitable for structural studies. The cysteine residue 110 was replaced with alanine, valine or serine. Protein secondary structure and stability (using circular dichroism spectropolarimetry), self-association (using analytical ultracentrifugation), and DNA-binding measurements revealed RTP.C110S to be the most similar mutant to wild-type RTP. The C110A and C110V.RTP mutants were less soluble, less stable and showed lower DNA-binding affinity. The structure of RTP.C110S, solved to 2.5A resolution using crystallographic methods, showed no major structural perturbation due to the mutation. Heteronuclear NMR spectroscopic studies revealed subtle differences in the electronic environment about the site of mutation. The study demonstrates the suitability of serine as a substitute for cysteine in RTP and the high sensitivity of protein behaviour to single amino acid substitutions.
