Evaluation of predictions of the stochastic model of organelle production based on exact distributions.

We present a reanalysis of the stochastic model of organelle production and show that the equilibrium distributions for the organelle numbers predicted by this model can be readily calculated in three different scenarios. These three distributions can be identified as standard distributions, and the corresponding exact formulae for their mean and variance can therefore be used in further analysis. This removes the need to rely on stochastic simulations or approximate formulae (derived using the fluctuation dissipation theorem). These calculations allow for further analysis of the predictions of the model. On the basis of this we question the extent to which the model can be used to conclude that peroxisome biogenesis is dominated by de novo production when Saccharomyces cerevisiae cells are grown on glucose medium.

Evaluation of predictions of the stochastic model of organelle production based on exact distributions.

We present a reanalysis of the stochastic model of organelle production and show that the equilibrium distributions for the organelle numbers predicted by this model can be readily calculated in three different scenarios. These three distributions can be identified as standard distributions, and the corresponding exact formulae for their mean and variance can therefore be used in further analysis. This removes the need to rely on stochastic simulations or approximate formulae (derived using the fluctuation dissipation theorem). These calculations allow for further analysis of the predictions of the model. On the basis of this we question the extent to which the model can be used to conclude that peroxisome biogenesis is dominated by de novo production when Saccharomyces cerevisiae cells are grown on glucose medium.

The spatial distribution of the exocyst and actin cortical patches is sufficient to organize hyphal tip growth.

In the hyphal tip of Candida albicans we have made detailed quantitative measurements of (i) exocyst components, (ii) Rho1, the regulatory subunit of (1,3)-ß-glucan synthase, (iii) Rom2, the specialized guanine-nucleotide exchange factor (GEF) of Rho1, and (iv) actin cortical patches, the sites of endocytosis. We use the resulting data to construct and test a quantitative 3-dimensional model of fungal hyphal growth based on the proposition that vesicles fuse with the hyphal tip at a rate determined by the local density of exocyst components. Enzymes such as (1,3)-ß-glucan synthase thus embedded in the plasma membrane continue to synthesize the cell wall until they are removed by endocytosis. The model successfully predicts the shape and dimensions of the hyphae, provided that endocytosis acts to remove cell wall-synthesizing enzymes at the subapical bands of actin patches. Moreover, a key prediction of the model is that the distribution of the synthase is substantially broader than the area occupied by the exocyst. This prediction is borne out by our quantitative measurements. Thus, although the model highlights detailed issues that require further investigation, in general terms the pattern of tip growth of fungal hyphae can be satisfactorily explained by a simple but quantitative model rooted within the known molecular processes of polarized growth. Moreover, the methodology can be readily adapted to model other forms of polarized growth, such as that which occurs in plant pollen tubes.

Reprogramming of Escherichia coli K-12 metabolism during the initial phase of transition from an anaerobic to a micro-aerobic environment.

BACKGROUND: Many bacteria undergo transitions between environments with differing O2 availabilities as part of their natural lifestyles and during biotechnological processes. However, the dynamics of adaptation when bacteria experience changes in O2 availability are understudied. The model bacterium and facultative anaerobe Escherichia coli K-12 provides an ideal system for exploring this process. METHODS AND FINDINGS: Time-resolved transcript profiles of E. coli K-12 during the initial phase of transition from anaerobic to micro-aerobic conditions revealed a reprogramming of gene expression consistent with a switch from fermentative to respiratory metabolism. The changes in transcript abundance were matched by changes in the abundances of selected central metabolic proteins. A probabilistic state space model was used to infer the activities of two key regulators, FNR (O2 sensing) and PdhR (pyruvate sensing). The model implied that both regulators were rapidly inactivated during the transition from an anaerobic to a micro-aerobic environment. Analysis of the external metabolome and protein levels suggested that the cultures transit through different physiological states during the process of adaptation, characterized by the rapid inactivation of pyruvate formate-lyase (PFL), a slower induction of pyruvate dehydrogenase complex (PDHC) activity and transient excretion of pyruvate, consistent with the predicted inactivation of PdhR and FNR. CONCLUSION: Perturbation of anaerobic steady-state cultures by introduction of a limited supply of O2 combined with time-resolved transcript, protein and metabolite profiling, and probabilistic modeling has revealed that pyruvate (sensed by PdhR) is a key metabolic signal in coordinating the reprogramming of E. coli K-12 gene expression by working alongside the O2 sensor FNR during transition from anaerobic to micro-aerobic conditions.

Distant cousins: genomic and sequence diversity within the BPI fold-containing (BPIF)/PLUNC protein family.

PLUNC (palate, lung and nasal epithelium clone) proteins make up the largest branch of the BPI (bactericidal/permeability-increasing protein)/LBP (lipopolysaccharide-binding protein) family of lipid-transfer proteins. PLUNCs make up one of the most rapidly evolving mammalian protein families and exhibit low levels of sequence similarity coupled with multiple examples of species-specific gene acquisition and gene loss. Vertebrate genomes contain multiple examples of genes that do not meet our original definition of what is required to be a member of the PLUNC family, namely conservation of exon numbers/sizes, overall protein size, genomic location and the presence of a conserved disulfide bond. This suggests that evolutionary forces have continued to act on the structure of this conserved domain in what are likely to be functionally important ways.

Systematic nomenclature for the PLUNC/PSP/BSP30/SMGB proteins as a subfamily of the BPI fold-containing superfamily.

We present the BPIFAn/BPIFBn systematic nomenclature for the PLUNC (palate lung and nasal epithelium clone)/PSP (parotid secretory protein)/BSP30 (bovine salivary protein 30)/SMGB (submandibular gland protein B) family of proteins, based on an adaptation of the SPLUNCn (short PLUNCn)/LPLUNCn (large PLUNCn) nomenclature. The nomenclature is applied to a set of 102 sequences which we believe represent the current reliable data for BPIFA/BPIFB proteins across all species, including marsupials and birds. The nomenclature will be implemented by the HGNC (HUGO Gene Nomenclature Committee).

The virulence factor PEB4 (Cj0596) and the periplasmic protein Cj1289 are two structurally related SurA-like chaperones in the human pathogen Campylobacter jejuni.

The PEB4 protein is an antigenic virulence factor implicated in host cell adhesion, invasion, and colonization in the food-borne pathogen Campylobacter jejuni. peb4 mutants have defects in outer membrane protein assembly and PEB4 is thought to act as a periplasmic chaperone. The crystallographic structure of PEB4 at 2.2-Å resolution reveals a dimer with distinct SurA-like chaperone and peptidyl-prolyl cis/trans isomerase (PPIase) domains encasing a large central cavity. Unlike SurA, the chaperone domain is formed by interlocking helices from each monomer, creating a domain-swapped architecture. PEB4 stimulated the rate of proline isomerization limited refolding of denatured RNase T(1) in a juglone-sensitive manner, consistent with parvulin-like PPIase domains. Refolding and aggregation of denatured rhodanese was significantly retarded in the presence of PEB4 or of an engineered variant specifically lacking the PPIase domain, suggesting the chaperone domain possesses a holdase activity. Using bioinformatics approaches, we identified two other SurA-like proteins (Cj1289 and Cj0694) in C. jejuni. The 2.3-Å structure of Cj1289 does not have the domain-swapped architecture of PEB4 and thus more resembles SurA. Purified Cj1289 also enhanced RNase T(1) refolding, although poorly compared with PEB4, but did not retard the refolding of denatured rhodanese. Structurally, Cj1289 is the most similar protein to SurA in C. jejuni, whereas PEB4 has most structural similarity to the Par27 protein of Bordetella pertussis. Our analysis predicts that Cj0694 is equivalent to the membrane-anchored chaperone PpiD. These results provide the first structural insights into the periplasmic assembly of outer membrane proteins in C. jejuni.

Identification and characterisation of the BPI/LBP/PLUNC-like gene repertoire in chickens reveals the absence of a LBP gene.

Palate, lung and nasal epithelial clone (PLUNC) proteins are structural homologues to the innate defence molecules LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI). PLUNCs make up the largest portion of the wider BPI/LBP/PLUNC-like protein family and are amongst the most rapidly evolving mammalian genes. In this study we systematically identified and characterised BPI/LBP/PLUNC-like protein-encoding genes in the chicken genome. We identified eleven complete genes (and a pseudogene). Five of them are clustered on a >50 kb locus on chromosome 20, immediately adjacent to BPI. In addition to BPI, we have identified presumptive orthologues LPLUNCs 2, 3, 4 and 6, and BPIL-2. We find no evidence for the existence of single domain containing proteins in birds. Strikingly our analysis also suggests that there is no LBP orthologue in chicken. This observation may in part account for the relative resistance to LPS toxicity observed in birds. Our results indicate significant differences between the avian and mammalian repertoires of BPI/LBP/PLUNC-like genes at the genomic and transcriptional levels and provide a framework for further functional analyses of this gene family in chickens.

When simple sequence comparison fails: the cryptic case of the shared domains of the bacterial replication initiation proteins DnaB and DnaD.

DnaD and DnaB are essential DNA-replication-initiation proteins in low-G+C content Gram-positive bacteria. Here we use sensitive Hidden Markov Model-based techniques to show that the DnaB and DnaD proteins share a common structure that is evident across all their structural domains, termed DDBH1 and DDBH2 (DnaD DnaB Homology 1 and 2). Despite strong sequence divergence, many of the DNA-binding and oligomerization properties of these domains have been conserved. Although eluding simple sequence comparisons, the DDBH2 domains share the only strong sequence motif; an extremely highly conserved YxxxIxxxW sequence that contributes to DNA binding. Sequence alignments of DnaD alone fail to identify another key part of the DNA-binding module, since it includes a poorly conserved sequence, a solvent-exposed and somewhat unstable helix and a mobile segment. We show by NMR, in vitro mutagenesis and in vivo complementation experiments that the DNA-binding module of Bacillus subtilis DnaD comprises the YxxxIxxxW motif, the unstable helix and a portion of the mobile region, the latter two being essential for viability. These structural insights lead us to a re-evaluation of the oligomerization and DNA-binding properties of the DnaD and DnaB proteins.

Dimerization of protein G B1 domain at low pH: a conformational switch caused by loss of a single hydrogen bond.

A number of signals in the NMR spectrum of the B1 domain of staphylococcal protein G (GB1) show a chemical shift dependence on the concentration of the protein at pH 3 but not at neutral pH, implying the existence of self-association at low pH. NMR backbone relaxation experiments show that GB1 undergoes a slow conformational exchange at pH 3, which is not seen at higher pH. Analysis of relaxation dispersion experiments yields a self-association constant of 50 mM, and shows that (15)N chemical shift changes in the dimer interface are up to 3 ppm. The shift changes measured from concentration-dependent HSQC spectra and from relaxation dispersion show good consistency. Measurements of chemical shifts as a function of pH show that a hydrogen bond between the sidechains of Asp44 and Gln40 is broken when Asp44 is protonated, and that loss of this hydrogen bond leads to the breaking of the (i, i + 4) backbone helical hydrogen bond from Asp44 HN to Gln40 O, and therefore to a loss of two residues from the C-terminal end of the helix. This weakens the helix structure and facilitates the loss of further helical structure thus permitting dimerization, which is suggested to occur in the same way as observed for the A42F mutant of GB1 (Jee et al., Proteins 2007;71:1420-1431), by formation of an antiparallel beta-sheet between the edge strands 2 in two monomers. The monomer/dimer ratio is thus a finely balanced equilibrium even in the wild type protein.

Dimerisation of the UBA domain of p62 inhibits ubiquitin binding and regulates NF-kappaB signalling.

The ubiquitin (Ub)-binding p62 scaffold protein (encoded by the SQSTM1 gene) regulates a diverse range of signalling pathways leading to activation of the nuclear factor kappa B (NF-kappaB) family of transcription factors and is an important regulator of macroautophagy. Mutations within the gene encoding p62 are commonly found in patients with Paget's disease of bone and largely cluster within the C-terminal ubiquitin-associated (UBA) domain, impairing its ability to bind Ub, resulting in dysregulated NF-kappaB signalling. However, precisely how Ub-binding is regulated at the molecular level is unclear. NMR relaxation dispersion experiments, coupled with concentration-dependent NMR, CD, isothermal titration calorimetry and fluorescence kinetic measurements, reveal that the p62 UBA domain forms a highly stable dimer (K(dim) approximately 4-12 microM at 298 K). NMR analysis shows that the dimer interface partially occludes the Ub-binding surface, particularly at the C-terminus of helix 3, making UBA dimerisation and Ub-binding mutually exclusive processes. Somewhat unusually, the monomeric UBA appears to be the biologically active form and the dimer appears to be the inactive one. Engineered point mutations in loop 1 (E409K and G410K) are shown to destabilise the dimer interface, lead to a higher proportion of the bound monomer and, in NF-kappaB luciferase reporter assays, are associated with reduced NF-kappaB activity compared with wt-p62.

Structural tightening and interdomain communication in the catalytic cycle of phosphoglycerate kinase.

Changes in amide-NH chemical shift and hydrogen exchange rates as phosphoglycerate kinase progresses through its catalytic cycle have been measured to assess whether they correlate with changes in hydrogen bonding within the protein. Four representative states were compared: the free enzyme, a product complex containing 3-phosphoglyceric acid (3PG), a substrate complex containing ADP and a transition-state analogue (TSA) complex containing a 3PG-AlF(4)(-)-ADP moiety. There are an overall increases in amide protection from hydrogen exchange when the protein binds the substrate and product ligands and an additional increase when the TSA complex is formed. This is consistent with stabilisation of the protein structure by ligand binding. However, there is no correlation between the chemical shift changes and the protection factor changes, indicating that the protection factor changes are not associated with an overall shortening of hydrogen bonds in the protected ground state, but rather can be ascribed to the properties of the high-energy, exchange-competent state. Therefore, an overall structural tightening mechanism is not supported by the data. Instead, we observed that some cooperativity is exhibited in the N-domain, such that within this domain the changes induced upon forming the TSA complex are an intensification of those induced by binding 3PG. Furthermore, chemical shift changes induced by 3PG binding extend through the interdomain region to the C-domain beta-sheet, highlighting a network of hydrogen bonds between the domains that suggests interdomain communication. Interdomain communication is also indicated by amide protection in one domain being significantly altered by binding of substrate to the other, even where no associated change in the structure of the substrate-free domain is indicated by chemical shifts. Hence, the communication between domains is also manifested in the accessibility of higher-energy, exchange-competent states. Overall, the data that are consistent with structural tightening relate to defined regions and are close to the 3PG binding site and in the hinge regions of 3-phosphoglycerate kinase.

Automated protein structure calculation from NMR data.

Current software is almost at the stage to permit completely automatic structure determination of small proteins of <15 kDa, from NMR spectra to structure validation with minimal user interaction. This goal is welcome, as it makes structure calculation more objective and therefore more easily validated, without any loss in the quality of the structures generated. Moreover, it releases expert spectroscopists to carry out research that cannot be automated. It should not take much further effort to extend automation to ca 20 kDa. However, there are technological barriers to further automation, of which the biggest are identified as: routines for peak picking; adoption and sharing of a common framework for structure calculation, including the assembly of an automated and trusted package for structure validation; and sample preparation, particularly for larger proteins. These barriers should be the main target for development of methodology for protein structure determination, particularly by structural genomics consortia.

Catalysis by Glomerella cingulata cutinase requires conformational cycling between the active and inactive states of its catalytic triad.

Cutinase belongs to a group of enzymes that catalyze the hydrolysis of esters and triglycerides. Structural studies on the enzyme from Fusarium solani have revealed the presence of a classic catalytic triad that has been implicated in the enzyme's mechanism. We have solved the crystal structure of Glomerella cingulata cutinase in the absence and in the presence of the inhibitors E600 (diethyl p-nitrophenyl phosphate) and PETFP (3-phenethylthio-1,1,1-trifluoropropan-2-one) to resolutions between 2.6 and 1.9 A. Analysis of these structures reveals that the catalytic triad (Ser136, Asp191, and His204) adopts an unusual configuration with the putative essential histidine His204 swung out of the active site into a position where it is unable to participate in catalysis, with the imidazole ring 11 A away from its expected position. Solution-state NMR experiments are consistent with the disrupted configuration of the triad observed crystallographically. H204N, a site-directed mutant, was shown to be catalytically inactive, confirming the importance of this residue in the enzyme mechanism. These findings suggest that, during its catalytic cycle, cutinase undergoes a significant conformational rearrangement converting the loop bearing the histidine from an inactive conformation, in which the histidine of the triad is solvent exposed, to an active conformation, in which the triad assumes a classic configuration.

The denatured state of N-PGK is compact and predominantly disordered.

The organisation of the structure present in the chemically denatured N-terminal domain of phosphoglycerate kinase (N-PGK) has been determined by paramagnetic relaxation enhancements (PREs) to define the conformational landscape accessible to the domain. Below 2.0 M guanidine hydrochloride (GuHCl), a species of N-PGK (denoted I(b)) is detected, distinct from those previously characterised by kinetic experiments [folded (F), kinetic intermediate (I(k)) and denatured (D)]. The transition to I(b) is never completed at equilibrium, because F predominates below 1.0 M GuHCl. Therefore, the ability of PREs to report on transient or low population species has been exploited to characterise I(b). Five single cysteine variants of N-PGK were labelled with the nitroxide electron spin-label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate] and the denaturant dependences of the relaxation properties of the amide NMR signals between 1.2 and 3.6 M GuHCl were determined. Significant PREs for I(b) were obtained, but these were distributed almost uniformly throughout the sequence. Furthermore, the PREs indicate that no specific short tertiary contacts persist. The data indicate a collapsed state with no coherent three-dimensional structure, but with a restricted radius beyond which the protein chain rarely reaches. The NMR characteristics of I(b) indicate that it forms from the fully denatured state within 100 micros, and therefore a rapid collapse is the initial stage of folding of N-PGK from its chemically denatured state. By extrapolation, I(b) is the predominant form of the denatured state under native conditions, and the non-specifically collapsed structure implies that many non-native contacts and chain reversals form early in protein folding and must be broken prior to attaining the native state topology.

Crystallization and preliminary X-ray analysis of recombinant Glomerella cingulata cutinase.

Cutinase catalyzes the hydrolysis of water-soluble esters and long-chain triglycerides and belongs to the family of serine hydrolases. The enzyme is thought to represent an evolutionary link between the esterase and lipase families and has potential applications in a wide range of industrial hydrolytic processes, for which an understanding of the molecular basis of its substrate specificity is critical. Glomerella cingulata cutinase has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a wide range of different crystal forms in the presence and absence of inhibitors. The best crystals are those of the apo cutinase, which diffract to beyond 1.6 A resolution and belong to space group P4(1)2(1)2 or P4(3)2(1)2. Crystals of cutinase with the inhibitors PETFP or E600 belong to space groups P2(1)2(1)2(1) and P2(1), respectively, and diffract to approximately 2.5 A resolution. All of the crystals are suitable for structural studies, which are currently ongoing.

Exclusion of the native alpha-helix from the amyloid fibrils of a mixed alpha/beta protein.

Members of the cystatin superfamily are involved in an inherited form of cerebral amyloid angiopathy and readily form amyloid fibrils in vitro. We have determined the structured core of human stefin B (cystatin B) amyloid fibrils using quenched hydrogen exchange and NMR. The core contains residues from four of the five strands of the native beta-sheet, delimited by unprotected loop regions analogous to those of the native monomeric structure. However, non-native features are also apparent, the most striking of which is the exclusion of the native alpha-helix. Before forming amyloid in vitro, cystatins dimerise via 3D domain swapping, and assemble into tetramers with trans to cis isomerism of a conserved proline. In the fibril, the hinge loop that forms an extended beta-structure in the dimer remains protected, consistent with the domain-swapping interface being maintained. However, the fibril data are not compatible with a simple 3D domain-swapping model for amyloid formation, and the displacement of the helix points to alternative packing arrangements of native-like beta-structure, in which proline isomerism is important in preventing steric clashing.

Structural and functional analysis of RNA and TAP binding to SF2/ASF.

The serine/arginine-rich (SR) protein splicing factor 2/alternative splicing factor (SF2/ASF) has a role in splicing, stability, export and translation of messenger RNA. Here, we present the structure of the RNA recognition motif (RRM) 2 from SF2/ASF, which has an RRM fold with a considerably extended loop 5 region, containing a two-stranded beta-sheet. The loop 5 extension places the previously identified SR protein kinase 1 docking sequence largely within the RRM fold. We show that RRM2 binds to RNA in a new way, by using a tryptophan within a conserved SWQLKD motif that resides on helix alpha1, together with amino acids from strand beta2 and a histidine on loop 5. The linker connecting RRM1 and RRM2 contains arginine residues, which provide a binding site for the mRNA export factor TAP, and when TAP binds to this region it displaces RNA bound to RRM2.

A thiol labelling competition experiment as a probe for sidechain packing in the kinetic folding intermediate of N-PGK.

Protein folding is directed by the sequence of sidechains along the polypeptide backbone, but despite this the developement of sidechain interactions during folding is not well understood. Here, the thiol-active reagent, dithio-nitrobenzoic acid (DTNB), is used to probe the exposure of the cysteine sidechain thiols in the kinetic folding intermediates of the N-terminal domain of phosphoglycerate kinase (N-PGK) and a number of conservative (I-, L-, or V-to-C) single cysteine variants. Rapid dilution of chemically denatured protein into folding conditions in the presence of DTNB allowed the degree of sidechain protection in any rapidly formed intermediate to be determined through the analysis of the kinetics of labelling. The protection factors derived for the intermediate(s) were generally small (<25), indicating only partial burial of the sidechains. The distribution of protection parallels the previously reported backbone amide protection for the folding intermediate of N-PGK. These observations are consistent with the hypothesis that such intermediates resemble molten globule states; i.e. with native-like backbone hydrogen bonding and overall tertiary structure, but with the sidechains that make up the hydrophobic protein core dynamic and intermittently solvent exposed. The success of the competition technique in characterizing this kinetic intermediate invites application to other model systems.