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The function of protein, RNA, and DNA is modulated by fast, dynamic exchanges between three-dimensional conformations. Conformational sampling of biomolecules with exact and nullspace inverse kinematics, using rotatable bonds as revolute joints and non-covalent interactions as holonomic constraints, can accurately characterize these native ensembles. However, sampling biomolecules remains challenging owing to their ultra-high dimensional configuration spaces, and the requirement to avoid (self-) collisions, which results in low acceptance rates. Here, we present two novel mechanisms to overcome these limitations. First, we introduced temporary constraints between near-colliding links. The resulting constraint varieties instantaneously redirect the search for collision-free conformations, and couple motions between distant parts of the linkage. Second, we adapted a randomized Poisson-disk motion planner, which prevents local oversampling and widens the search, to ultra-high dimensions. We evaluated our algorithm on several model systems. Our contributions apply to general high-dimensional motion planning problems in static and dynamic environments with obstacles., 15 pages, 5 figures, submitted to WAFR16

Noncoding ribonucleic acids (RNA) play a critical role in a wide variety of cellular processes, ranging from regulating gene expression to post-translational modification and protein synthesis. Their activity is modulated by highly dynamic exchanges between three-dimensional conformational substates, which are difficult to characterize experimentally and computationally. Here, we present an innovative, entirely kinematic computational procedure to efficiently explore the native ensemble of RNA molecules. Our procedure projects degrees of freedom onto a subspace of conformation space defined by distance constraints in the tertiary structure. The dimensionality reduction enables efficient exploration of conformational space. We show that the conformational distributions obtained with our method broadly sample the conformational landscape observed in NMR experiments. Compared to normal mode analysis-based exploration, our procedure diffuses faster through the experimental ensemble while also accessing conformational substates to greater precision. Our results suggest that conformational sampling with a highly reduced but fully atomistic representation of noncoding RNA expresses key features of their dynamic nature.

Sufu (Suppressor of Fused), a two-domain protein, plays a critical role in regulating Hedgehog signaling and is conserved from flies to humans. A few bacterial Sufu-like proteins have previously been identified based on sequence similarity to the N-terminal domain of eukaryotic Sufu proteins, but none have been structurally or biochemically characterized and their function in bacteria is unknown. We have determined the crystal structure of a more distantly related Sufu-like homolog, NGO1391 from Neisseria gonorrhoeae, at 1.4 A resolution, which provides the first biophysical characterization of a bacterial Sufu-like protein. The structure revealed a striking similarity to the N-terminal domain of human Sufu (r.m.s.d. of 2.6 A over 93% of the NGO1391 protein), despite an extremely low sequence identity of ∼15%. Subsequent sequence analysis revealed that NGO1391 defines a new subset of smaller, Sufu-like proteins that are present in ∼200 bacterial species and has resulted in expansion of the SUFU (PF05076) family in Pfam.

The crystal structure of BT_3984, a SusD-family protein, reveals a TPR N-terminal region providing support for a loop-rich C-terminal subdomain and suggests possible interfaces involved in sus complex formation., The crystal structure of the Bacteroides thetaiotaomicron protein BT_3984 was determined to a resolution of 1.7 Å and was the first structure to be determined from the extensive SusD family of polysaccharide-binding proteins. SusD is an essential component of the sus operon that defines the paradigm for glycan utilization in dominant members of the human gut microbiota. Structural analysis of BT_3984 revealed an N-terminal region containing several tetratricopeptide repeats (TPRs), while the signature C-terminal region is less structured and contains extensive loop regions. Sequence and structure analysis of BT_3984 suggests the presence of binding interfaces for other proteins from the polysaccharide-utilization complex.

The crystal structure of BT2081 from B. thetaiotaomicron reveals a two-domain protein with a putative carbohydrate-binding site in the C-­terminal domain., BT2081 from Bacteroides thetaiotaomicron (GenBank accession code NP_810994.1) is a member of a novel protein family consisting of over 160 members, most of which are found in the different classes of Bacteroidetes. Genome-context analysis lends support to the involvement of this family in carbohydrate metabolism, which plays a key role in B. thetaiotaomicron as a predominant bacterial symbiont in the human distal gut microbiome. The crystal structure of BT2081 at 2.05 Å resolution represents the first structure from this new protein family. BT2081 consists of an N-terminal domain, which adopts a β-sandwich immunoglobulin-like fold, and a larger C-terminal domain with a β-sandwich jelly-roll fold. Structural analyses reveal that both domains are similar to those found in various carbohydrate-active enzymes. The C-terminal β-jelly-roll domain contains a potential carbohydrate-binding site that is highly conserved among BT2081 homologs and is situated in the same location as the carbohydrate-binding sites that are found in structurally similar glycoside hydrolases (GHs). However, in BT2081 this site is partially occluded by surrounding loops, which results in a deep solvent-accessible pocket rather than a shallower solvent-exposed cleft.

The first structures from the FmdE Pfam family (PF02663) reveal that some members of this family form tightly intertwined dimers consisting of two domains (N-terminal α+β core and C-terminal zinc-finger domains), whereas others contain only the core domain. The presence of the zinc-finger domain suggests that some members of this family may perform functions associated with transcriptional regulation, protein–protein interaction, RNA binding or metal-ion sensing., Examination of the genomic context for members of the FmdE Pfam family (PF02663), such as the protein encoded by the fmdE gene from the methanogenic archaeon Methanobacterium thermoautotrophicum, indicates that 13 of them are co-transcribed with genes encoding subunits of molybdenum formylmethanofuran dehydrogenase (EC 1.2.99.5), an enzyme that is involved in microbial methane production. Here, the first crystal structures from PF02663 are described, representing two bacterial and one archaeal species: B8FYU2_DESHY from the anaerobic dehalogenating bacterium Desulfito­bacterium hafniense DCB-2, Q2LQ23_SYNAS from the syntrophic bacterium Syntrophus aciditrophicus SB and Q9HJ63_THEAC from the thermoacidophilic archaeon Thermoplasma acidophilum. Two of these proteins, Q9HJ63_THEAC and Q2LQ23_SYNAS, contain two domains: an N-terminal thioredoxin-like α+β core domain (NTD) consisting of a five-stranded, mixed β-sheet flanked by several α-helices and a C-terminal zinc-finger domain (CTD). B8FYU2_DESHY, on the other hand, is composed solely of the NTD. The CTD of Q9HJ63_THEAC and Q2LQ23_SYNAS is best characterized as a treble-clef zinc finger. Two significant structural differences between Q9HJ63_THEAC and Q2LQ23_SYNAS involve their metal binding. First, zinc is bound to the putative active site on the NTD of Q9HJ63_THEAC, but is absent from the NTD of Q2LQ23_SYNAS. Second, whereas the structure of the CTD of Q2LQ23_SYNAS shows four Cys side chains within coordination distance of the Zn atom, the structure of Q9HJ63_THEAC is atypical for a treble-cleft zinc finger in that three Cys side chains and an Asp side chain are within coordination distance of the zinc.

The crystal structure of the prephenate dehydrogenase component of the bifunctional H. influenzae TyrA reveals unique structural differences between bifunctional and monofunctional TyrA enzymes., Chorismate mutase/prephenate dehydrogenase from Haemophilus influenzae Rd KW20 is a bifunctional enzyme that catalyzes the rearrangement of chorismate to prephenate and the NAD(P)+-dependent oxidative decarboxyl­ation of prephenate to 4-hydroxyphenylpyruvate in tyrosine biosynthesis. The crystal structure of the prephenate dehydrogenase component (HinfPDH) of the TyrA protein from H. influenzae Rd KW20 in complex with the inhibitor tyrosine and cofactor NAD+ has been determined to 2.0 Å resolution. HinfPDH is a dimeric enzyme, with each monomer consisting of an N-terminal α/β dinucleotide-binding domain and a C-terminal α-helical dimerization domain. The structure reveals key active-site residues at the domain interface, including His200, Arg297 and Ser179 that are involved in catalysis and/or ligand binding and are highly conserved in TyrA proteins from all three kingdoms of life. Tyrosine is bound directly at the catalytic site, suggesting that it is a competitive inhibitor of HinfPDH. Comparisons with its structural homologues reveal important differences around the active site, including the absence of an α–β motif in HinfPDH that is present in other TyrA proteins, such as Synechocystis sp. arogenate dehydrogenase. Residues from this motif are involved in discrimination between NADP+ and NAD+. The loop between β5 and β6 in the N-terminal domain is much shorter in HinfPDH and an extra helix is present at the C-terminus. Furthermore, HinfPDH adopts a more closed conformation compared with TyrA proteins that do not have tyrosine bound. This conformational change brings the substrate, cofactor and active-site residues into close proximity for catalysis. An ionic network consisting of Arg297 (a key residue for tyrosine binding), a water molecule, Asp206 (from the loop between β5 and β6) and Arg365′ (from the additional C-terminal helix of the adjacent monomer) is observed that might be involved in gating the active site.

The crystal structure of a novel MACPF protein, which may play a role in the adaptation of commensal bacteria to host environments in the human gut, was determined and analyzed., Membrane-attack complex/perforin (MACPF) proteins are transmembrane pore-forming proteins that are important in both human immunity and the virulence of pathogens. Bacterial MACPFs are found in diverse bacterial species, including most human gut-associated Bacteroides species. The crystal structure of a bacterial MACPF-domain-containing protein BT_3439 (Bth-MACPF) from B. thetaiotaomicron, a predominant member of the mammalian intestinal microbiota, has been determined. Bth-MACPF contains a membrane-attack complex/perforin (MACPF) domain and two novel C-terminal domains that resemble ribonuclease H and interleukin 8, respectively. The entire protein adopts a flat crescent shape, characteristic of other MACPF proteins, that may be important for oligomerization. This Bth-MACPF structure provides new features and insights not observed in two previous MACPF structures. Genomic context analysis infers that Bth-MACPF may be involved in a novel protein-transport or nutrient-uptake system, suggesting an important role for these MACPF proteins, which were likely to have been inherited from eukaryotes via horizontal gene transfer, in the adaptation of commensal bacteria to the host environment.

The crystal structure of BT1062 from Bacteroides thetaiotaomicron revealed a conserved fold that is widely adopted by fimbrial components., BT1062 from Bacteroides thetaiotaomicron is a homolog of Mfa2 (PGN0288 or PG0179), which is a component of the minor fimbriae in Porphyromonas gingivalis. The crystal structure of BT1062 revealed a conserved fold that is widely adopted by fimbrial components.

Mre11 nuclease plays a central role in the repair of cytotoxic and mutagenic DNA double-strand breaks. As X-ray structural information has been available only for the Pyrococcus furiosus enzyme (PfMre11), the conserved and variable features of this nuclease across the domains of life have not been experimentally defined. Our crystal structure and biochemical studies demonstrate that TM1635 from Thermotoga maritima, originally annotated as a putative nuclease, is an Mre11 endo/exonuclease (TmMre11) and the first such structure from eubacteria. TmMre11 and PfMre11 display similar overall structures, despite sequence identity in the twilight zone of only approximately 20%. However, they differ substantially in their DNA-specificity domains and in their dimeric organization. Residues in the nuclease domain are highly conserved, but those in the DNA-specificity domain are not. The structural differences likely affect how Mre11 from different organisms recognize and interact with single-stranded DNA, double-stranded DNA and DNA hairpin structures during DNA repair. The TmMre11 nuclease active site has no bound metal ions, but is conserved in sequence and structure with the exception of a histidine that is important in PfMre11 nuclease activity. Nevertheless, biochemical characterization confirms that TmMre11 possesses both endonuclease and exonuclease activities on single-stranded and double-stranded DNA substrates, respectively.

The crystal structure of SSO2064, the first structural representative of Pfam family PF01796 (DUF35), reveals a two-domain architecture comprising an N-terminal zinc-ribbon domain and a C-terminal OB-fold domain. Analysis of the domain architecture, operon organization and bacterial orthologs combined with the structural features of SSO2064 suggests a role involving acyl-CoA binding for this family of proteins., SSO2064 is the first structural representative of PF01796 (DUF35), a large prokaryotic family with a wide phylogenetic distribution. The structure reveals a novel two-domain architecture comprising an N-terminal, rubredoxin-like, zinc ribbon and a C-terminal, oligonucleotide/oligosaccharide-binding (OB) fold domain. Additional N-terminal helical segments may be involved in protein–protein interactions. Domain architectures, genomic context analysis and functional evidence from certain bacterial representatives of this family suggest that these proteins form a novel fatty-acid-binding component that is involved in the biosynthesis of lipids and polyketide antibiotics and that they possibly function as acyl-CoA-binding proteins. This structure has led to a re-evaluation of the DUF35 family, which has now been split into two entries in the latest Pfam release (v.24.0).

The crystal structures of SPO0140 and Sbal_2486 revealed a two-domain structure that adopts a novel fold. Analysis of the interdomain cleft suggests a nucleotide-based ligand with a genome context indicating signaling as a possible role for this family., The crystal structures of SPO0140 and Sbal_2486 were determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). The structures revealed a conserved core with domain duplication and a superficial similarity of the C-terminal domain to pleckstrin homology-like folds. The conservation of the domain interface indicates a potential binding site that is likely to involve a nucleotide-based ligand, with genome-context and gene-fusion analyses additionally supporting a role for this family in signal transduction, possibly during oxidative stress.

Surface layers (S-layers) are 2D, proteinaceous lattices that form the outermost cell envelope component of many microbes. S-layers, which exhibit exceptional sequence diversity, are found in nearly all archaea and numerous bacteria. Despite their variation, S-layer proteins display several unifying characteristics such as their ability to form crystalline sheets punctuated with nano-scale pores, and their propensity for charged amino acids. However, the precise role of these charged functional groups and how they relate to cellular function is unknown. Here, we offer a rationale for charged S-layer proteins in the context of the structural evolution of S-layers. We have chosen ammonia-oxidizing archaea (AOA) as a model system for S-layer and used the cryo electron tomographic reconstruction of the AOA to develop a 2D electro-diffusion reaction computational framework to simulate diffusion and consumption of the charged solute ammonium. The AOA create energy directly from electrons evolved during ammonia oxidation by ammonia monooxygenase (AMO). While the specific location of the archaeal AMO active site is unknown, a bacterial homologue indicates a location underneath the S-layer in pseudo periplasmic space (PPS). Our simulations suggest that charged S-layers and nanopores expedite diffusion of charged solutes into the PPS when the electro-diffusion-reaction system is driven away from equilibrium, replenishing reacted NH_4^+ in PPS. By contrast, a neutral S-layer would inhibit diffusion of charged molecules, while removing the S-layer altogether dramatically reduced ammonium concentration throughout the PPS. Strikingly, analysis of annotated S-layer amino acid sequences from all known archaeal clades indicated a clear dearth of sequences in the neutral regime. Our simulations suggest that charged S-layers and nanopores impart a potential fitness advantage. Thus, S-layer charge may have emerged by convergent evolution to enhance metabolic function in diverse ecosystems.

The crystal structure of the NGO1945 gene product from N. gonorrhoeae (UniProt Q5F5IO) reveals that the N-terminal domain assigned as a domain of unknown function (DUF2063) is likely to bind DNA and that the protein may be involved in transcriptional regulation., Proteins with the DUF2063 domain constitute a new Pfam family, PF09836. The crystal structure of a member of this family, NGO1945 from Neisseria gonorrhoeae, has been determined and reveals that the N-terminal DUF2063 domain is likely to be a DNA-binding domain. In conjunction with the rest of the protein, NGO1945 is likely to be involved in transcriptional regulation, which is consistent with genomic neighborhood analysis. Of the 216 currently known proteins that contain a DUF2063 domain, the most significant sequence homologs of NGO1945 (∼40–99% sequence identity) are from various Neisseria and Haemophilus species. As these are important human pathogens, NGO1945 represents an interesting candidate for further exploration via biochemical studies and possible therapeutic intervention.

The first structural representative of the PF08866 (DUF1831) protein family reveals a potential new α+β fold and indicates a possible involvement in amino-acid metabolism., The structure of LP2179, a member of the PF08866 (DUF1831) family, suggests a novel α+β fold comprising two β-sheets packed against a single helix. A remote structural similarity to two other uncharacterized protein families specific to the Bacillus genus (PF08868 and PF08968), as well as to prokaryotic S-adenosylmethionine decarboxylases, is consistent with a role in amino-acid metabolism. Genomic neighborhood analysis of LP2179 supports this functional assignment, which might also then be extended to PF08868 and PF08968.

KPN03535 is a protein unique to K. pneumoniae. The crystal structure reveals that KPN03535 represents a novel variant of the OB-fold and is likely to be a DNA-binding lipoprotein., KPN03535 (gi|152972051) is a putative lipoprotein of unknown function that is secreted by Klebsiella pneumoniae MGH 78578. The crystal structure reveals that despite a lack of any detectable sequence similarity to known structures, it is a novel variant of the OB-fold and structurally similar to the bacterial Cpx-pathway protein NlpE, single-stranded DNA-binding (SSB) proteins and toxins. K. pneumoniae MGH 78578 forms part of the normal human skin, mouth and gut flora and is an opportunistic pathogen that is linked to about 8% of all hospital-acquired infections in the USA. This structure provides the foundation for further investigations into this divergent member of the OB-fold family.

PA1994, a Pfam PF06475 (DUF1089) family homolog from P. aeruginosa, reveals remote similarities to lipoprotein localization factors and a conserved putative glycolipid-binding site., The crystal structure of PA1994 from Pseudomonas aeruginosa, a member of the Pfam PF06475 family classified as a domain of unknown function (DUF1089), reveals a novel fold comprising a 15-stranded β-sheet wrapped around a single α-helix that assembles into a tight dimeric arrangement. The remote structural similarity to lipoprotein localization factors, in addition to the presence of an acidic pocket that is conserved in DUF1089 homologs, phospholipid-binding and sugar-binding proteins, indicate a role for PA1994 and the DUF1089 family in glycolipid metabolism. Genome-context analysis lends further support to the involvement of this family of proteins in glycolipid metabolism and indicates possible activation of DUF1089 homologs under conditions of bacterial cell-wall stress or host–pathogen interactions.

Cell cycle regulated stalk biogenesis in Caulobacter crescentus is controlled by a multi-step phosphorelay system consisting of the hybrid histidine kinase ShkA, the histidine-phosphotransfer protein ShpA and the response regulator TacA. ShpA shuttles phosphoryl groups between ShkA and TacA. When phosphorylated, TacA triggers a downstream transcription cascade for stalk synthesis in an RpoN-dependent manner. The crystal structure of ShpA was determined to 1.52 Å resolution. ShpA belongs to a family of monomeric histidine phosphotransfer (HPt) proteins, which feature a highly conserved four-helix bundle. The phosphorylatable histidine, His56, is located on the surface of the helix bundle and is fully solvent exposed. One end of the four-helix bundle in ShpA is shorter compared to other characterized histidine phosphotransfer proteins, whereas the face that potentially interacts with the response regulators is structurally conserved. Similarities of the interaction surface around the phosphorylation site suggest that ShpA is likely to share a common mechanism for molecular recognition and phosphotransfer with yeast phosphotransfer protein YPD1 despite low overall sequence similarity.

ECX21941 represents a very large family (over 600 members) of novel, ocean metagenome-specific proteins identified by clustering of the dataset from the Global Ocean Sampling expedition. The crystal structure of ECX21941 reveals unexpected similarity to Sm/LSm proteins, which are important RNA-binding proteins, despite no detectable sequence similarity. The ECX21941 protein assembles as a homopentamer in solution and in the crystal structure when expressed in Escherichia coli and represents the first pentameric structure for this Sm/LSm family of proteins, although the actual oligomeric form in vivo is currently not known. The genomic neighborhood analysis of ECX21941 and its homologs combined with sequence similarity searches suggest a cyanophage origin for this protein. The specific functions of members of this family are unknown, but our structure analysis of ECX21941 indicates nucleic acid-binding capabilities and suggests a role in RNA and/or DNA processing.

The protein SO4266 (gi|24375750) from the bacterium Shewanella oneidensis MR-1 is annotated as a member of Pfam PF02661. This family consists of Fic (filamentation induced by cAMP) proteins and their relatives, and is characterized by the presence of a well-conserved HPFXXGNG motif 1. The biochemistry of Fic proteins has not been characterized extensively and their exact molecular functions remain unknown. From early studies in Escherichia coli, it is believed that Fic proteins and cAMP may be involved in a regulatory mechanism of cell division, including folate metabolism by the synthesis of p-aminobenzoic acid (PABA) or folate 1. Proteins containing the Fic domain are present in all kingdoms of life and range in size from ~200 to 500 amino acids. The Fic protein family contains 647 members, including two human proteins, according to Pfam (May 2008). Sequence-based clustering 2 of this protein family, at 30% sequence identity, groups these proteins into 18 clusters. Three crystal structures of Fic proteins from bacteria (unpublished) are available in the Protein Data Bank [accession codes 2g03 (194 residues, 2.2 A), 2f6s (201 residues, 2.5 A) and 3cuc (262 residues, 2.7 A)]. The first two of these proteins belong to a single cluster of 16 members and share ~60% sequence identity. The anti-apoptotic bacterial effector protein BepA, which is a type IV secretion (T4S) system substrate, also contains an N-terminal Fic domain 3. In humans, the Fic domain is present in the Huntingtin Interacting Protein E (HYPE; Uniprot entry Q9BVA6_HUMAN), a protein of unknown function that is thought to interact with Huntingtin, one of the major proteins in the Huntington's disease protein interaction network (listed as NAD- or FAD-binding) 4. Bioinformatics analysis of prokaryotic toxin-antitoxin networks 5 suggests that Fic proteins are putative death-on-curing (Doc) toxins that are part of the Phd-Doc system. These proteins likely function as metal-dependent nucleases or RNA-processing enzymes, 5 while more recent studies suggest that Doc toxicity is caused by inhibition of translation elongation 6. SO4266 (Uniprot entry Q8E9K5_SHEON), at 372 amino acids, is one of the largest Fic domain-containing proteins to have its structure determined. Interestingly, both HYPE and SO4266 belong to the largest sequence cluster in this family (n.b. our B. thetaiotaomicron {"type":"entrez-protein","attrs":{"text":"NP_811426.1","term_id":"29347923","term_text":"NP_811426.1"}}NP_811426.1 structure with PDB id 3cuc also belongs to this cluster), which comprises 466 out of 647 proteins, and share ~32% sequence identity in the Fic domain. Here, we report the crystal structure of the SO4266 protein at 1.6 A resolution. The structure reveals a dimeric protein with additional electron density in the vicinity of the highly conserved HPFXXGNG motif in the Fic domain of one subunit that corresponds to the N-terminus of a symmetry-related molecule. In addition, the study also reveals a C-terminal winged-helix DNA-binding domain that sets it apart from the other Fic protein structures. The structure presented here is a representative of the largest sequence cluster and together with the structures of the other Fic proteins paves the way for further structure-based functional characterization.

Signal transduction ATPases with numerous domains (STAND), a large class of P-loop NTPases, belong to AAA+ ATPases. They include AP(apoptotic)-ATPases (e.g., animal apoptosis regulators CED4/Apaf-1, plant disease resistance proteins, and bacterial AfsR-like transcription regulators), NACHT NTPases (e.g. CARD4, NAIP, Het-E-1, TLP1), and several other less well-characterized families. STAND differ from other P-loop NTPases by their unique sequence motifs, which include an hhGRExE (h, hydrophobic; x, any residue) motif at the N-terminal region, a GxP/GxxP motif at the C-terminal region of the NTPase domain, in addition to a C-terminal helical domain and additional domains such as WD40, TPR, LRR or catalytic modules. Despite significant biological interests, structural coverage of STAND proteins is very limited and only two other structures are currently known: the cell death regulators Apaf-1 and CED-4. Here, we report the crystal structure of SSO1545 from Sulfolobus solfataricus, which was determined using the semi-automated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG; http://www.jcsg.org), as part of the National Institute of General Medical Sciences' Protein Structure Initiative (PSI). SSO1545 (NP-342973.1), a representative of the archaeal STANDs, is a member of Pfam PF01637 and encodes a protein of 356 residues with calculated molecular weight and isoelectric point of 41.7more » kD and 8.2, respectively.« less

Several applications in biology—e.g., incorporation of protein flexibility in ligand docking algorithms, interpretation of fuzzy X-ray crystallographic data, and homology modeling—require computing the internal parameters of a flexible fragment (usually, a loop) of a protein in order to connect its termini to the rest of the protein without causing any steric clash inside the loop and with the rest of the protein. One must often sample many such conformations in order to explore and adequately represent the conformational range of the studied loop. While sampling must be fast, it is made difficult by the fact that two conflicting constraints—kinematic closure and clash avoidance—must be satisfied concurrently. This paper describes two efficient and complementary sampling algorithms to explore the space of closed clash-free conformations of a flexible protein loop. The “seed sampling” algorithm samples broadly from this space, while the “deformation sampling” algorithm uses seed conformations as starting points to explore the conformation space around them at a finer grain. Computational results are presented for various loops ranging from 5 to 25 residues. More specific results also show that the combination of the sampling algorithms with a functional site prediction software (FEATURE) makes it possible to compute and recognize calcium-binding loop conformations. The sampling algorithms are implemented in a toolkit, called LoopTK, which is available at https://simtk.org/home/looptk.

To extend the structural coverage of proteins with unknown functions, we targeted a novel protein family (Pfam accession number PF08807, DUF1798) for which we proposed and determined the structures of two representative members. The MW1337R gene of Staphylococcus aureus subsp. aureus Rosenbach (Wood 46) encodes a protein with a molecular weight of 13.8 kDa (residues 1-116) and a calculated isoelectric point of 5.15. The lin2004 gene of the nonspore-forming bacterium Listeria innocua Clip11262 encodes a protein with a molecular weight of 14.6 kDa (residues 1-121) and a calculated isoelectric point of 5.45. MW1337R and lin2004, as well as their homologs, which, so far, have been found only in Bacillus, Staphylococcus, Listeria, and related genera (Geobacillus, Exiguobacterium, and Oceanobacillus), have unknown functions and are annotated as hypothetical proteins. The genomic contexts of MW1337R and lin2004 are similar and conserved in related species. In prokaryotic genomes, most often, functionally interacting proteins are coded by genes, which are colocated in conserved operons. Proteins from the same operon as MW1337R and lin2004 either have unknown functions (i.e., belong to DUF1273, Pfam accession number PF06908) or are similar to ypsB from Bacillus subtilis. The function of ypsB is unclear, although it has a strong similaritymore » to the N-terminal region of DivIVA, which was characterized as a bifunctional protein with distinct roles during vegetative growth and sporulation. In addition, members of the DUF1273 family display distant sequence similarity with the DprA/Smf protein, which acts downstream of the DNA uptake machinery, possibly in conjunction with RecA. The RecA activities in Bacillus subtilis are modulated by RecU Holliday-junction resolvase. In all analyzed cases, the gene coding for RecU is in the vicinity of MW1337R, lin2004, or their orthologs, but on a different operon located in the complementary DNA strand. Here, we report the crystal structures of MW1337R and lin2004, which were determined using the semiautomated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG), part of the National Institute of General Medical Sciences Protein Structure Initiative.« less

TyrA is a member of the dye-decolorizing peroxidase (DyP) family, a new family of heme-dependent peroxidase recently identified in fungi and bacteria. Here, we report the crystal structure of TyrA in complex with iron protoporphyrin (IX) at 2.3 A. TyrA is a dimer, with each monomer exhibiting a two-domain, alpha/beta ferredoxin-like fold. Both domains contribute to the heme-binding site. Co-crystallization in the presence of an excess of iron protoporphyrin (IX) chloride allowed for the unambiguous location of the active site and the specific residues involved in heme binding. The structure reveals a Fe-His-Asp triad essential for heme positioning, as well as a novel conformation of one of the heme propionate moieties compared to plant peroxidases. Structural comparison to the canonical DyP family member, DyP from Thanatephorus cucumeris (Dec 1), demonstrates conservation of this novel heme conformation, as well as residues important for heme binding. Structural comparisons with representative members from all classes of the plant, bacterial, and fungal peroxidase superfamily demonstrate that TyrA, and by extension the DyP family, adopts a fold different from all other structurally characterized heme peroxidases. We propose that a new superfamily be added to the peroxidase classification scheme to encompass the DyP family of heme peroxidases.

Calmodulin (CaM) is the primary calcium signaling protein in eukaryotes and has been extensively studied using various biophysical techniques. Prior crystal structures have noted the presence of ambiguous electron density in both hydrophobic binding pockets of Ca2+-CaM, but no assignment of these features has been made. In addition, Ca2+-CaM samples many conformational substates in the crystal and accurately modeling the full range of this functionally important disorder is challenging. In order to characterize these features in a minimally biased manner, a 1.0 Å resolution single-wavelength anomalous diffraction data set was measured for selenomethionine-substituted Ca2+-CaM. Density-modified electron-density maps enabled the accurate assignment of Ca2+-CaM main-chain and side-chain disorder. These experimental maps also substantiate complex disorder models that were automatically built using low-contour features of model-phased electron density. Furthermore, experimental electron-density maps reveal that 2-methyl-2,4-pentanediol (MPD) is present in the C-terminal domain, mediates a lattice contact between N-terminal domains and may occupy the N-terminal binding pocket. The majority of the crystal structures of target-free Ca2+-CaM have been derived from crystals grown using MPD as a precipitant, and thus MPD is likely to be bound in functionally critical regions of Ca2+-CaM in most of these structures. The adventitious binding of MPD helps to explain differences between the Ca2+-CaM crystal and solution structures and is likely to favor more open conformations of the EF-hands in the crystal.

Proteins operate and interact with partners by dynamically exchanging between functional substates of a conformational ensemble on a rugged free energy landscape. Understanding how these substates are linked by coordinated, collective motions requires exploring a high-dimensional space, which remains a tremendous challenge. While molecular dynamics simulations can provide atomically detailed insight into the dynamics, computational demands to adequately sample conformational ensembles of large biomolecules and their complexes often require tremendous resources. Kinematic models can provide high-level insights into conformational ensembles and molecular rigidity beyond the reach of molecular dynamics by reducing the dimensionality of the search space. Here, we model a protein as a kinematic linkage and present a new geometric method to characterize molecular rigidity from the constraint manifold Q and its tangent space T q Q at the current configuration q. In contrast to methods based on combinatorial constraint counting, our method is valid for both generic and non-generic, e.g., singular configurations. Importantly, our geometric approach provides an explicit basis for collective motions along floppy modes, resulting in an efficient procedure to probe conformational space. An atomically detailed structural characterization of coordinated, collective motions would allow us to engineer or allosterically modulate biomolecules by selectively stabilizing conformations that enhance or inhibit function with broad implications for human health.

Biomolecules adopt a dynamic ensemble of conformations, each with the potential to interact with binding partners or perform the chemical reactions required for a multitude of cellular functions. Recent advances in X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, and other techniques are helping us realize the dream of seeing—in atomic detail—how different parts of biomolecules exchange between functional sub-states using concerted motions. Integrative structural biology has advanced our understanding of the formation of large macromolecular complexes and how their components interact in assemblies by leveraging data from many low-resolution methods. Here, we review the growing opportunities for integrative, dynamic structural biology at the atomic scale, contending there is increasing synergistic potential between X-ray crystallography, NMR, and computer simulations to reveal a structural basis for protein conformational dynamics at high resolution.

Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free electron laser (XFEL) crystallography. The “diffraction-before-destruction” nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states for a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.

A new software package,DISTL(Diffraction Image Screening Tool and Library), for the rapid analysis of X-ray diffraction patterns collected from macromolecular crystals is presented. Within seconds, the program characterizes the strength and quality of the Bragg spots, determines the limiting resolution of the image, and identifies deleterious features such as ice-rings and intense salt reflections. The procedure also generates a reliable set of intense peaks for auto-indexing. The ability to classify a large number of crystals quickly will be especially useful at synchrotron and home-laboratory X-ray sources where automated crystal screening and data collection systems have been implemented.