Progress in low-resolution ab initio phasing with CrowdPhase.

Ab initio phasing by direct computational methods in low-resolution X-ray crystallography is a long-standing challenge. A common approach is to consider it as two subproblems: sampling of phase space and identification of the correct solution. While the former is amenable to a myriad of search algorithms, devising a reliable target function for the latter problem remains an open question. Here, recent developments in CrowdPhase, a collaborative online game powered by a genetic algorithm that evolves an initial population of individuals with random genetic make-up (i.e. random phases) each expressing a phenotype in the form of an electron-density map, are presented. Success relies on the ability of human players to visually evaluate the quality of these maps and, following a Darwinian survival-of-the-fittest concept, direct the search towards optimal solutions. While an initial study demonstrated the feasibility of the approach, some important crystallographic issues were overlooked for the sake of simplicity. To address these, the new CrowdPhase includes consideration of space-group symmetry, a method for handling missing amplitudes, the use of a map correlation coefficient as a quality metric and a solvent-flattening step. Performances of this installment are discussed for two low-resolution test cases based on bona fide diffraction data.

The PduL Phosphotransacylase Is Used To Recycle Coenzyme A within the Pdu Microcompartment.

UNLABELLED: In Salmonella enterica, 1,2-propanediol (1,2-PD) utilization (Pdu) is mediated by a bacterial microcompartment (MCP). The Pdu MCP consists of a multiprotein shell that encapsulates enzymes and cofactors for 1,2-PD catabolism, and its role is to sequester a reactive intermediate (propionaldehyde) to minimize cellular toxicity and DNA damage. For the Pdu MCP to function, the enzymes encapsulated within must be provided with a steady supply of substrates and cofactors. In the present study, Western blotting assays were used to demonstrate that the PduL phosphotransacylase is a component of the Pdu MCP. We also show that the N-terminal 20-residue-long peptide of PduL is necessary and sufficient for targeting PduL and enhanced green fluorescent protein (eGFP) to the lumen of the Pdu MCP. We present the results of genetic tests that indicate that PduL plays a role in the recycling of coenzyme A internally within the Pdu MCP. However, the results indicate that some coenzyme A recycling occurs externally to the Pdu MCP. Hence, our results support a model in which a steady supply of coenzyme A is provided to MCP lumen enzymes by internal recycling by PduL as well as by the movement of coenzyme A across the shell by an unknown mechanism. These studies expand our understanding of the Pdu MCP, which has been linked to enteric pathogenesis and which provides a possible basis for the development of intracellular bioreactors for use in biotechnology. IMPORTANCE: Bacterial MCPs are widespread organelles that play important roles in pathogenesis and global carbon fixation. Here we show that the PduL phosphotransacylase is a component of the Pdu MCP. We also show that PduL plays a key role in cofactor homeostasis by recycling coenzyme A internally within the Pdu MCP. Further, we identify a potential N-terminal targeting sequence using a bioinformatic approach and show that this short sequence extension is necessary and sufficient for directing PduL as well as heterologous proteins to the lumen of the Pdu MCP. These findings expand our general understanding of bacterial MCP assembly and cofactor homeostasis.

Exploring bacterial organelle interactomes: a model of the protein-protein interaction network in the Pdu microcompartment.

Bacterial microcompartments (MCPs) are protein-bound organelles that carry out diverse metabolic pathways in a wide range of bacteria. These supramolecular assemblies consist of a thin outer protein shell, reminiscent of a viral capsid, which encapsulates sequentially acting enzymes. The most complex MCP elucidated so far is the propanediol utilizing (Pdu) microcompartment. It contains the reactions for degrading 1,2-propanediol. While several experimental studies on the Pdu system have provided hints about its organization, a clear picture of how all the individual components interact has not emerged yet. Here we use co-evolution-based methods, involving pairwise comparisons of protein phylogenetic trees, to predict the protein-protein interaction (PPI) network governing the assembly of the Pdu MCP. We propose a model of the Pdu interactome, from which selected PPIs are further inspected via computational docking simulations. We find that shell protein PduA is able to serve as a "universal hub" for targeting an array of enzymes presenting special N-terminal extensions, namely PduC, D, E, L and P. The varied N-terminal peptides are predicted to bind in the same cleft on the presumptive luminal face of the PduA hexamer. We also propose that PduV, a protein of unknown function with remote homology to the Ras-like GTPase superfamily, is likely to localize outside the MCP, interacting with the protruding ß-barrel of the hexameric PduU shell protein. Preliminary experiments involving a bacterial two-hybrid assay are presented that corroborate the existence of a PduU-PduV interaction. This first systematic computational study aimed at characterizing the interactome of a bacterial microcompartment provides fresh insight into the organization of the Pdu MCP.

Identification of a unique Fe-S cluster binding site in a glycyl-radical type microcompartment shell protein.

Recently, progress has been made toward understanding the functional diversity of bacterial microcompartment (MCP) systems, which serve as protein-based metabolic organelles in diverse microbes. New types of MCPs have been identified, including the glycyl-radical propanediol (Grp) MCP. Within these elaborate protein complexes, BMC-domain shell proteins [bacterial microcompartment (in reference to the shell protein domain)] assemble to form a polyhedral barrier that encapsulates the enzymatic contents of the MCP. Interestingly, the Grp MCP contains a number of shell proteins with unusual sequence features. GrpU is one such shell protein whose amino acid sequence is particularly divergent from other members of the BMC-domain superfamily of proteins that effectively defines all MCPs. Expression, purification, and subsequent characterization of the protein showed, unexpectedly, that it binds an iron-sulfur cluster. We determined X-ray crystal structures of two GrpU orthologs, providing the first structural insight into the homohexameric BMC-domain shell proteins of the Grp system. The X-ray structures of GrpU, both obtained in the apo form, combined with spectroscopic analyses and computational modeling, show that the metal cluster resides in the central pore of the BMC shell protein at a position of broken 6-fold symmetry. The result is a structurally polymorphic iron-sulfur cluster binding site that appears to be unique among metalloproteins studied to date.

CrowdPhase: crowdsourcing the phase problem.

The human mind innately excels at some complex tasks that are difficult to solve using computers alone. For complex problems amenable to parallelization, strategies can be developed to exploit human intelligence in a collective form: such approaches are sometimes referred to as `crowdsourcing'. Here, a first attempt at a crowdsourced approach for low-resolution ab initio phasing in macromolecular crystallography is proposed. A collaborative online game named CrowdPhase was designed, which relies on a human-powered genetic algorithm, where players control the selection mechanism during the evolutionary process. The algorithm starts from a population of `individuals', each with a random genetic makeup, in this case a map prepared from a random set of phases, and tries to cause the population to evolve towards individuals with better phases based on Darwinian survival of the fittest. Players apply their pattern-recognition capabilities to evaluate the electron-density maps generated from these sets of phases and to select the fittest individuals. A user-friendly interface, a training stage and a competitive scoring system foster a network of well trained players who can guide the genetic algorithm towards better solutions from generation to generation via gameplay. CrowdPhase was applied to two synthetic low-resolution phasing puzzles and it was shown that players could successfully obtain phase sets in the 30° phase error range and corresponding molecular envelopes showing agreement with the low-resolution models. The successful preliminary studies suggest that with further development the crowdsourcing approach could fill a gap in current crystallographic methods by making it possible to extract meaningful information in cases where limited resolution might otherwise prevent initial phasing.

Structure of dihydromethanopterin reductase, a cubic protein cage for redox transfer.

Dihydromethanopterin reductase (Dmr) is a redox enzyme that plays a key role in generating tetrahydromethanopterin (H4MPT) for use in one-carbon metabolism by archaea and some bacteria. DmrB is a bacterial enzyme understood to reduce dihydromethanopterin (H2MPT) to H4MPT using flavins as the source of reducing equivalents, but the mechanistic details have not been elucidated previously. Here we report the crystal structure of DmrB from Burkholderia xenovorans at a resolution of 1.9 Å. Unexpectedly, the biological unit is a 24-mer composed of eight homotrimers located at the corners of a cubic cage-like structure. Within a homotrimer, each monomer-monomer interface exhibits an active site with two adjacently bound flavin mononucleotide (FMN) ligands, one deeply buried and tightly bound and one more peripheral, for a total of 48 ligands in the biological unit. Computational docking suggested that the peripheral site could bind either the observed FMN (the electron donor for the overall reaction) or the pterin, H2MPT (the electron acceptor for the overall reaction), in configurations ideal for electron transfer to and from the tightly bound FMN. On this basis, we propose that DmrB uses a ping-pong mechanism to transfer reducing equivalents from FMN to the pterin substrate. Sequence comparisons suggested that the catalytic mechanism is conserved among the bacterial homologs of DmrB and partially conserved in archaeal homologs, where an alternate electron donor is likely used. In addition to the mechanistic revelations, the structure of DmrB could help guide the development of anti-obesity drugs based on modification of the ecology of the human gut.

The shells of BMC-type microcompartment organelles in bacteria.

Bacterial microcompartments are large proteinaceous structures that act as metabolic organelles in many bacterial cells. A shell or capsid, which is composed of a few thousand protein subunits, surrounds a series of sequentially acting enzymes and controls the diffusion of substrates and products into and out of the lumen. The carboxysome and the propanediol utilization microcompartment represent two well-studied systems among seven or more distinct types that can be delineated presently. Recent structural studies have highlighted a number of sophisticated mechanisms that underlie the function of bacterial microcompartment shell proteins. This review updates our understanding of bacterial microcompartment shells, how they are assembled, and how they carry out their functions in molecular transport and enzyme organization.

Using comparative genomics to uncover new kinds of protein-based metabolic organelles in bacteria.

Bacterial microcompartment (MCP) organelles are cytosolic, polyhedral structures consisting of a thin protein shell and a series of encapsulated, sequentially acting enzymes. To date, different microcompartments carrying out three distinct types of metabolic processes have been characterized experimentally in various bacteria. In the present work, we use comparative genomics to explore the existence of yet uncharacterized microcompartments encapsulating a broader set of metabolic pathways. A clustering approach was used to group together enzymes that show a strong tendency to be encoded in chromosomal proximity to each other while also being near genes for microcompartment shell proteins. The results uncover new types of putative microcompartments, including one that appears to encapsulate B(12) -independent, glycyl radical-based degradation of 1,2-propanediol, and another potentially involved in amino alcohol metabolism in mycobacteria. Preliminary experiments show that an unusual shell protein encoded within the glycyl radical-based microcompartment binds an iron-sulfur cluster, hinting at complex mechanisms in this uncharacterized system. In addition, an examination of the computed microcompartment clusters suggests the existence of specific functional variations within certain types of MCPs, including the alpha carboxysome and the glycyl radical-based microcompartment. The findings lead to a deeper understanding of bacterial microcompartments and the pathways they sequester.

PRDB: Protein Repeat DataBase.

Rapidly increasing genomic data present new challenges for scientists: making sense of millions of amino acid sequences requires a systematic approach and information about their 3D structure, function, and evolution. Over the last decade, numerous studies demonstrated the fundamental importance of protein tandem repeats and their involvement in human diseases. Bioinformatics analysis of these regions requires special computer programs and databases, since the conventional approaches predominantly developed for globular domains have limited success. To perform a global comparative analysis of protein tandem repeats, we developed the Protein Tandem Repeat DataBase (PRDB). PRDB is a curated database that includes the protein tandem repeats found in sequence databanks by the T-REKS program. The database is available at http://bioinfo.montp.cnrs.fr/?r=repeatDB.

Widespread disulfide bonding in proteins from thermophilic archaea.

Disulfide bonds are generally not used to stabilize proteins in the cytosolic compartments of bacteria or eukaryotic cells, owing to the chemically reducing nature of those environments. In contrast, certain thermophilic archaea use disulfide bonding as a major mechanism for protein stabilization. Here, we provide a current survey of completely sequenced genomes, applying computational methods to estimate the use of disulfide bonding across the Archaea. Microbes belonging to the Crenarchaeal branch, which are essentially all hyperthermophilic, are universally rich in disulfide bonding while lesser degrees of disulfide bonding are found among the thermophilic Euryarchaea, excluding those that are methanogenic. The results help clarify which parts of the archaeal lineage are likely to yield more examples and additional specific data on protein disulfide bonding, as increasing genomic sequencing efforts are brought to bear.

Protein homorepeats sequences, structures, evolution, and functions.

The vast majority of protein sequences are aperiodic; they do not have any strong bias in the amino acid composition, and they use a subtle mixture of all or most of the 20 amino acid residues to code a great number of various structures and functions. In this context, homorepeats, runs of a single amino acid residue, represent unusual, eye-catching motifs in proteins. Despite the sequence simplicity and relatively small size, the homorepeat runs have a strong potential for molecular interactions due to the excessively high local concentration of a certain physico-chemical property. Appearance of such runs within proteins may give them new structural and functional features. An increasing number of studies demonstrate the abundance of these motifs in proteins, their important roles in biological processes, and their link to a number of hereditary and age-related diseases. In this chapter, we summarize data on the distribution of homorepeats in proteomes and on their structural properties, evolution, and functions.

Protein tandem repeats - the more perfect, the less structured.

We analysed the structural properties of protein regions containing arrays of perfect and nearly perfect tandem repeats. Naturally occurring proteins with perfect repeats are practically absent among the proteins with known 3D structures. The great majority of such regions in the Protein Data Bank are found in the proteins designed de novo. The abundance of natural structured proteins with tandem repeats is inversely correlated with the repeat perfection: the chance of finding natural structured proteins in the Protein Data Bank increases with a decrease in the level of repeat perfection. Prediction of intrinsic disorder within the tandem repeats in the SwissProt proteins supports the conclusion that the level of repeat perfection correlates with their tendency to be unstructured. This correlation is valid across the various species and subcellular localizations, although the level of disordered tandem repeats varies significantly between these datasets. On average, in prokaryotes, tandem repeats of cytoplasmic proteins were predicted to be the most structured, whereas in eukaryotes, the most structured portion of the repeats was found in the membrane proteins. Our study supports the hypothesis that, in general, the repeat perfection is a sign of recent evolutionary events rather than of exceptional structural and (or) functional importance of the repeat residues.

T-REKS: identification of Tandem REpeats in sequences with a K-meanS based algorithm.

MOTIVATION: Over the last years a number of evidences have been accumulated about high incidence of tandem repeats in proteins carrying fundamental biological functions and being related to a number of human diseases. At the same time, frequently, protein repeats are strongly degenerated during evolution and, therefore, cannot be easily identified. To solve this problem, several computer programs which were based on different algorithms have been developed. Nevertheless, our tests showed that there is still room for improvement of methods for accurate and rapid detection of tandem repeats in proteins. RESULTS: We developed a new program called T-REKS for ab initio identification of the tandem repeats. It is based on clustering of lengths between identical short strings by using a K-means algorithm. Benchmark of the existing programs and T-REKS on several sequence datasets is presented. Our program being linked to the Protein Repeat DataBase opens the way for large-scale analysis of protein tandem repeats. T-REKS can also be applied to the nucleotide sequences. AVAILABILITY: The algorithm has been implemented in JAVA, the program is available upon request at http://bioinfo.montp.cnrs.fr/?r=t-reks. Protein Repeat DataBase generated by using T-REKS is accessible at http://bioinfo.montp.cnrs.fr/?r=repeatDB.