ABSTRACT
Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli.
Subject(s)
Codon/genetics , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial/genetics , Protein Biosynthesis/genetics , RNA, Bacterial/metabolism , RNA, Messenger/metabolism , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/biosynthesis , Genes, Synthetic/genetics , Half-Life , Kinetics , Logistic Models , Models, Genetic , Molecular Sequence Data , Odds Ratio , Peptide Chain Elongation, Translational , RNA Folding , RNA Stability , RNA, Bacterial/genetics , RNA, Messenger/genetics , Transcription, Genetic/genetics , Viral Proteins/metabolismABSTRACT
Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design.
Subject(s)
Bacterial Proteins/chemistry , Carboxy-Lyases/chemistry , Mutation, Missense , Amino Acid Sequence , Carboxy-Lyases/deficiency , Carboxy-Lyases/genetics , Catalytic Domain , Crystallography, X-Ray , Deficiency Diseases/genetics , Enzyme Stability , Humans , Hydrogen Bonding , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Structure, Quaternary , Protein Structure, Secondary , Structural Homology, ProteinABSTRACT
Recent studies of signal transduction in bacteria have revealed a unique second messenger, bis-(3'-5')-cyclic dimeric GMP (c-di-GMP), which regulates transitions between motile states and sessile states, such as biofilms. C-di-GMP is synthesized from two GTP molecules by diguanylate cyclases (DGC). The catalytic activity of DGCs depends on a conserved GG(D/E)EF domain, usually part of a larger multi-domain protein organization. The domains other than the GG(D/E)EF domain often control DGC activation. This paper presents the 1.83 Å crystal structure of an isolated catalytically competent GG(D/E)EF domain from the A1U3W3_MARAV protein from Marinobacter aquaeolei. Co-crystallization with GTP resulted in enzymatic synthesis of c-di-GMP. Comparison with previously solved DGC structures shows a similar orientation of c-di-GMP bound to an allosteric regulatory site mediating feedback inhibition of the enzyme. Biosynthesis of c-di-GMP in the crystallization reaction establishes that the enzymatic activity of this DGC domain does not require interaction with regulatory domains.
Subject(s)
Bacterial Proteins/chemistry , Cyclic GMP/analogs & derivatives , Escherichia coli Proteins/chemistry , Marinobacter/chemistry , Phosphorus-Oxygen Lyases/chemistry , Allosteric Regulation , Allosteric Site , Amino Acid Sequence , Conserved Sequence , Crystallography, X-Ray/methods , Cyclic GMP/biosynthesis , Cyclic GMP/chemistry , Enzyme Activation , Guanosine Triphosphate/chemistry , Marinobacter/enzymology , Molecular Sequence Data , Protein Conformation , Protein Interaction Mapping , Protein Structure, Tertiary , Sequence Analysis, ProteinABSTRACT
The conserved Lipoprotein-17 domain of membrane-associated protein Q9PRA0_UREPA from Ureaplasma parvum was selected for structure determination by the Northeast Structural Genomics Consortium, as part of the Protein Structure Initiative's program on structure-function analysis of protein domains from large domain sequence families lacking structural representatives. The 100-residue Lipoprotein-17 domain is a "domain of unknown function" (DUF) that is a member of Pfam protein family PF04200, a large domain family for which no members have characterized biochemical functions. The three-dimensional structure of the Lipoprotein-17 domain of protein Q9PRA0_UREPA was determined by both solution NMR and by X-ray crystallography at 2.5 Å. The two structures are in good agreement with each other. The domain structure features three α-helices, α1 through α3, and five ß-strands. Strands ß1/ß2, ß3/ß4, ß4/ß5 are anti-parallel to each other. Strands ß1and ß2 are orthogonal to strands ß3, ß4, ß5, while helix α3 is formed between the strands ß3 and ß4. One-turn helix α2 is formed between the strands ß1 and ß2, while helix α1 occurs in the N-terminal polypeptide segment. Searches of the Protein Data Bank do not identify any other protein with significant structural similarity to Lipoprotein-17 domain of Q9PRA0_UREPA, indicating that it is a novel protein fold.
Subject(s)
Lipoproteins/chemistry , Membrane Proteins/chemistry , Nuclear Magnetic Resonance, Biomolecular , Ureaplasma/metabolism , Amino Acid Sequence , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Mycoplasma/metabolism , Protein Conformation , Protein Folding , Protein Structure, Tertiary , SolutionsABSTRACT
Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1826-1831). This reaction is formally identical to that catalyzed by MiaB on the C2 of adenosine 37 near the anticodon of several tRNAs. We present spectroscopic evidence that Thermotoga maritima RimO, like MiaB, contains two [4Fe-4S] centers, one presumably bound to three invariant cysteines in the central radical S-adenosylmethionine (AdoMet) domain and the other to three invariant cysteines in the N-terminal UPF0004 domain. We demonstrate that holo-RimO can specifically methylthiolate the aspartate residue of a 20-mer peptide derived from S12, yielding a mixture of mono- and bismethylthio derivatives. Finally, we present the 2.0 A crystal structure of the central radical AdoMet and the C-terminal TRAM (tRNA methyltransferase 2 and MiaB) domains in apo-RimO. Although the core of the open triose-phosphate isomerase (TIM) barrel of the radical AdoMet domain was conserved, RimO showed differences in domain organization compared with other radical AdoMet enzymes. The unusually acidic TRAM domain, likely to bind the basic S12 protein, is located at the distal edge of the radical AdoMet domain. The basic S12 protein substrate is likely to bind RimO through interactions with both the TRAM domain and the concave surface of the incomplete TIM barrel. These biophysical results provide a foundation for understanding the mechanism of methylthioation by radical AdoMet enzymes in the MiaB/RimO family.
Subject(s)
Protein Processing, Post-Translational/physiology , Ribosomal Proteins/metabolism , S-Adenosylmethionine/metabolism , Sulfurtransferases/metabolism , Thermotoga maritima/enzymology , Crystallography, X-Ray , Protein Structure, Tertiary , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/genetics , Structure-Activity Relationship , Sulfurtransferases/chemistry , Sulfurtransferases/genetics , Thermotoga maritima/geneticsABSTRACT
Crystallization is the most serious bottleneck in high-throughput protein-structure determination by diffraction methods. We have used data mining of the large-scale experimental results of the Northeast Structural Genomics Consortium and experimental folding studies to characterize the biophysical properties that control protein crystallization. This analysis leads to the conclusion that crystallization propensity depends primarily on the prevalence of well-ordered surface epitopes capable of mediating interprotein interactions and is not strongly influenced by overall thermodynamic stability. We identify specific sequence features that correlate with crystallization propensity and that can be used to estimate the crystallization probability of a given construct. Analyses of entire predicted proteomes demonstrate substantial differences in the amino acid-sequence properties of human versus eubacterial proteins, which likely reflect differences in biophysical properties, including crystallization propensity. Our thermodynamic measurements do not generally support previous claims regarding correlations between sequence properties and protein stability.
Subject(s)
Crystallization , Proteins/chemistry , Algorithms , Animals , Biophysics/methods , Computational Biology/methods , Entropy , Epitopes/chemistry , Humans , Models, Statistical , Protein Folding , Surface Properties , ThermodynamicsABSTRACT
The structure of a structural genomics target protein, Tbru020260AAA from Trypanosoma brucei, has been determined to a resolution of 2.2 A using multiple-wavelength anomalous diffraction at the Se K edge. This protein belongs to Pfam sequence family PF08538 and is only distantly related to previously studied members of the alpha/beta-hydrolase fold family. Structural superposition onto representative alpha/beta-hydrolase fold proteins of known function indicates that a possible catalytic nucleophile, Ser116 in the T. brucei protein, lies at the expected location. However, the present structure and by extension the other trypanosomatid members of this sequence family have neither sequence nor structural similarity at the location of other active-site residues typical for proteins with this fold. Together with the presence of an additional domain between strands beta6 and beta7 that is conserved in trypanosomatid genomes, this suggests that the function of these homologs has diverged from other members of the fold family.
Subject(s)
Hydrolases/chemistry , Protein Folding , Trypanosoma brucei brucei/enzymology , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , Dimerization , Hydrolases/isolation & purification , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Serine/chemistryABSTRACT
The second messenger cyclic diguanylate (c-di-GMP) controls the transition between motile and sessile growth in eubacteria, but little is known about the proteins that sense its concentration. Bioinformatics analyses suggested that PilZ domains bind c-di-GMP and allosterically modulate effector pathways. We have determined a 1.9 A crystal structure of c-di-GMP bound to VCA0042/PlzD, a PilZ domain-containing protein from Vibrio cholerae. Either this protein or another specific PilZ domain-containing protein is required for V. cholerae to efficiently infect mice. VCA0042/PlzD comprises a C-terminal PilZ domain plus an N-terminal domain with a similar beta-barrel fold. C-di-GMP contacts seven of the nine strongly conserved residues in the PilZ domain, including three in a seven-residue long N-terminal loop that undergoes a conformational switch as it wraps around c-di-GMP. This switch brings the PilZ domain into close apposition with the N-terminal domain, forming a new allosteric interaction surface that spans these domains and the c-di-GMP at their interface. The very small size of the N-terminal conformational switch is likely to explain the facile evolutionary diversification of the PilZ domain.
Subject(s)
Bacterial Proteins/chemistry , Cyclic GMP/analogs & derivatives , Protein Structure, Secondary , Protein Structure, Tertiary , Signal Transduction/physiology , Amino Acid Sequence , Animals , Bacterial Proteins/classification , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Cyclic GMP/chemistry , Cyclic GMP/genetics , Cyclic GMP/metabolism , Humans , Mice , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Phylogeny , Protein Binding , Protein Structure, Quaternary , Sequence Alignment , Sequence Homology, Amino Acid , Vibrio cholerae/metabolism , Vibrio cholerae/pathogenicityABSTRACT
The structure of a putative Raf kinase inhibitor protein (RKIP) homolog from the eukaryotic parasite Plasmodium vivax has been studied to a resolution of 1.3 A using multiple-wavelength anomalous diffraction at the Se K edge. This protozoan protein is topologically similar to previously studied members of the phosphatidylethanolamine-binding protein (PEBP) sequence family, but exhibits a distinctive left-handed alpha-helical region at one side of the canonical phospholipid-binding site. Re-examination of previously determined PEBP structures suggests that the P. vivax protein and yeast carboxypeptidase Y inhibitor may represent a structurally distinct subfamily of the diverse PEBP-sequence family.
Subject(s)
Phosphatidylethanolamine Binding Protein/chemistry , Phosphatidylethanolamine Binding Protein/physiology , Plasmodium vivax/chemistry , Amino Acid Motifs/physiology , Amino Acid Sequence , Animals , Binding Sites/physiology , Crystallography, X-Ray , Molecular Sequence Data , Phosphatidylethanolamine Binding Protein/metabolism , Plasmodium vivax/metabolism , Protein Conformation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Structure, Secondary/physiology , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Protozoan Proteins/physiologyABSTRACT
We report here the crystal structure at 2.0 A resolution of the AGR_C_4470p protein from the Gram-negative bacterium Agrobacterium tumefaciens. The protein is a tightly associated dimer, each subunit of which bears strong structural homology with the two domains of the heme utilization protein ChuS from Escherichia coli and HemS from Yersinia enterocolitica. Remarkably, the organization of the AGR_C_4470p dimer is the same as that of the two domains in ChuS and HemS, providing structural evidence that these two proteins evolved by gene duplication. However, the binding site for heme, while conserved in HemS and ChuS, is not conserved in AGR_C_4470p, suggesting that it probably has a different function. This is supported by the presence of two homologs of AGR_C_4470p in E. coli, in addition to the ChuS protein.
Subject(s)
Agrobacterium tumefaciens/metabolism , Bacterial Proteins/chemistry , Membrane Transport Proteins/chemistry , Oxidoreductases/chemistry , Amino Acid Sequence , Binding Sites , Conserved Sequence , Crystallography, X-Ray , Dimerization , Heme Oxygenase (Decyclizing)/chemistry , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence AlignmentABSTRACT
The 1.8 A resolution de novo structure of nucleoside 2-deoxyribosyltransferase (EC 2.4.2.6) from Trypanosoma brucei (TbNDRT) has been determined by SADa phasing in an unliganded state and several ligand-bound states. This enzyme is important in the salvage pathway of nucleoside recycling. To identify novel lead compounds, we exploited "fragment cocktail soaks". Out of 304 compounds tried in 31 cocktails, four compounds could be identified crystallographically in the active site. In addition, we demonstrated that very short soaks of approximately 10 s are sufficient even for rather hydrophobic ligands to bind in the active site groove, which is promising for the application of similar soaking experiments to less robust crystals of other proteins.