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1.  Crystal structure of O-methyltransferase CalO6 from the calicheamicin biosynthetic pathway: a case of challenging structure determination at low resolution 
Background
Calicheamicins (CAL) are enedyine natural products with potent antibiotic and cytotoxic activity, used in anticancer therapy. The O-methyltransferase CalO6 is proposed to catalyze methylation of the hydroxyl moiety at the C2 position of the orsellinic acid group of CAL.
Results
Crystals of CalO6 diffracted non-isotropically, with the usable data extending to 3.4 Å. While no single method of crystal structure determination yielded a structure of CalO6, we were able to determine its structure by using molecular replacement-guided single wavelength anomalous dispersion by using diffraction data from native crystals of CalO6 and a highly non-isomorphous mercury derivative. The structure of CalO6 reveals the methyltransferase fold and dimeric organization characteristic of small molecule O-methyltransferases involved in secondary metabolism in bacteria and plants. Uncommonly, CalO6 was crystallized in the absence of S-adenosylmethionine (SAM; the methyl donor) or S-adenosylhomocysteine (SAH; its product).
Conclusions
Likely as a consequence of the dynamic nature of CalO6 in the absence of its cofactor, the central region of CalO6, which forms a helical lid-like structure near the active site in CalO6 and similar enzymes, is not observed in the electron density. We propose that this region controls the entry of SAM into and the exit of SAH from the active site of CalO6 and shapes the active site for substrate binding and catalysis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12900-015-0040-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12900-015-0040-6
PMCID: PMC4501126  PMID: 26170207
Anticancer drug; Enediyne; Low-resolution refinement; Methylation; Natural product biosynthesis
2.  Structure of the stationary phase survival protein YuiC from B.subtilis 
Background
Stationary phase survival proteins (Sps) were found in Firmicutes as having analogous domain compositions, and in some cases genome context, as the resuscitation promoting factors of Actinobacteria, but with a different putative peptidoglycan cleaving domain.
Results
The first structure of a Firmicute Sps protein YuiC from B. subtilis, is found to be a stripped down version of the cell-wall peptidoglycan hydrolase MltA. The YuiC structures are of a domain swapped dimer, although some monomer is also found in solution. The protein crystallised in the presence of pentasaccharide shows a 1,6-anhydrodisaccharide sugar product, indicating that YuiC cleaves the sugar backbone to form an anhydro product at least on lengthy incubation during crystallisation.
Conclusions
The structural simplification of MltA in Sps proteins is analogous to that of the resuscitation promoting factor domains of Actinobacteria, which are stripped down versions of lysozyme and soluble lytic transglycosylase proteins.
Electronic supplementary material
The online version of this article (doi:10.1186/s12900-015-0039-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12900-015-0039-z
PMCID: PMC4499186  PMID: 26163297
Dormancy; Stationary phase survival; Peptidoglycan; Resuscitation promoting factors; Firmicutes
3.  Variations in periplasmic loop interactions determine the pH-dependent activity of the hexameric urea transporter UreI from Helicobacter pylori: a molecular dynamics study 
Background
Helicobacter pylori is an important factor in the development of diseases such as ulcer and gastric cancer. This bacterium uses a periplasmic transporter, UreI, to deliver urea to the intracelullar space, where later it is transformed into ammonia by the cytoplasmic enzyme urease to survive the acidic condition of the human stomach. The UreI transporter presents a pH-dependent activity, where this pH-dependence remains unknown at a structural level. Althought the existance of several protonable residues in the periplasmic loops are related to the pH-dependent activity, we find interesting to have a clear view of the conformational changes involved in this phenomena through a molecular dynamic study.
Results
Molecular dynamic simulations of the UreI transporter at three different pH conditions were performed, revealing two main pH-dependent conformations, which we present as the open and close states. We find that salt bridges between the periplasmic loops are crucial interactions that stabilize these conformations. Besides, a cooperative behaviour exists between the six subunits of the system that is necessary to fulfill the activity of this transporter.
Conclusions
We found different pH-dependent conformations of the urea transporter UreI from Helicobacter pylori, which are related to salt-bridge interactions in the periplasmic regions. The behaviour of every channel in the system is not independent, given the existance of a cooperative behaviour through the formation of salt-bridges between the subunits of the hexameric system. We believe that our results will be related to the generation of new eradication therapies using this transporter as an attractive target, denoting that the knowledge of the possible pH-dependent conformations adopted for this transporter are important for the development of rational drug design approximations.
Electronic supplementary material
The online version of this article (doi:10.1186/s12900-015-0038-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12900-015-0038-0
PMCID: PMC4482100  PMID: 26112768
Helicobacter pylori; UreI; pH; Molecular dynamics; Periplasmic loop
4.  Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA Glycosylase 
Background
Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized.
Results
This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This also represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. Comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms.
Conclusion
The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.
Electronic supplementary material
The online version of this article (doi:10.1186/s12900-015-0037-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s12900-015-0037-1
PMCID: PMC4450493  PMID: 26031450
Protein-DNA structure; Non-specific DNA; Early DNA recognition complex; Uracil-DNA glycosylase; Poxvirus
5.  Modeling of the OX1R–orexin-A complex suggests two alternative binding modes 
Background
Interactions between the orexin peptides and their cognate OX1 and OX2 receptors remain poorly characterized. Site-directed mutagenesis studies on orexin peptides and receptors have indicated amino acids important for ligand binding and receptor activation. However, a better understanding of specific pairwise interactions would benefit small molecule discovery.
Results
We constructed a set of three-dimensional models of the orexin 1 receptor based on the 3D-structures of the orexin 2 receptor (released while this manuscript was under review), neurotensin receptor 1 and chemokine receptor CXCR4, conducted an exhaustive docking of orexin-A16–33 peptide fragment with ZDOCK and RDOCK, and analyzed a total of 4301 complexes through multidimensional scaling and clustering. The best docking poses reveal two alternative binding modes, where the C-terminus of the peptide lies deep in the binding pocket, on average about 5–6 Å above Tyr6.48 and close to Gln3.32. The binding modes differ in the about 100° rotation of the peptide; the peptide His26 faces either the receptor’s fifth transmembrane helix or the seventh helix. Both binding modes are well in line with previous mutation studies and partake in hydrogen bonding similar to suvorexant.
Conclusions
We present two binding modes for orexin-A into orexin 1 receptor, which help rationalize previous results from site-directed mutagenesis studies. The binding modes should serve small molecule discovery, and offer insights into the mechanism of receptor activation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12900-015-0036-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12900-015-0036-2
PMCID: PMC4469407  PMID: 25957175
Orexin-A; OX1 receptor; Peptide docking; G protein-coupled receptor; Pose selection; Multidimensional scaling; GPCR
6.  Human coronavirus OC43 3CL protease and the potential of ML188 as a broad-spectrum lead compound: Homology modelling and molecular dynamic studies 
Background
The coronavirus 3 chymotrypsin-like protease (3CLpro) is a validated target in the design of potential anticoronavirus inhibitors. The high degree of homology within the protease’s active site and substrate conservation supports the identification of broad spectrum lead compounds. A previous study identified the compound ML188, also termed 16R, as an inhibitor of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) 3CLpro. This study will detail the generation of a homology model of the 3CLpro of the human coronavirus OC43 and determine the potential of 16R to form a broad-spectrum lead compound. MODELLER was used to generate a suitable three-dimensional model of the OC43 3CLpro and the Prime module of Schrӧdinger predicted the binding conformation and free energy of binding of 16R within the 3CLpro active site. Molecular dynamics further confirmed ligand stability and hydrogen bonding networks.
Results
A high quality homology model of the OC43 3CLpro was successfully generated in an active conformation. Further studies reproduced the binding pose of 16R within the active site of the generated model, where its free energy of binding was shown to equal that of the 3CLpro of SARS-CoV, a receptor it is experimentally proven to inhibit. The stability of the ligand was subsequently confirmed by molecular dynamics.
Conclusion
The lead compound 16R may represent a broad-spectrum inhibitor of the 3CLpro of OC43 and potentially other coronaviruses. This study provides an atomistic structure of the 3CLpro of OC43 and supports further experimental validation of the inhibitory effects of 16R. These findings further confirm that the 3CLpro of coronaviruses can be inhibited by broad spectrum lead compounds.
doi:10.1186/s12900-015-0035-3
PMCID: PMC4411765  PMID: 25928480
Human coronavirus; OC43; 3CLpro; Homology modelling; Molecular dynamics
7.  Computational re-engineering of Amylin sequence with reduced amyloidogenic potential 
Background
The aggregation of amyloid proteins into fibrils is associated with neurodegenerative diseases such as Alzheimer’s and Type II Diabetes. Different methods have explored ways to impede and inhibit amyloid aggregation. Most attempts in the literature involve applying stress to the environment around amyloids. Varying pH levels, modifying temperature, applying pressure through protein crowding and ligand docking are classical examples of these methods. However, environmental stress usually affects molecular pathways and protein functions in the cell and is challenging to construct in vivo. In this paper, we explore destabilizing amyloid proteins through the manipulation of genetic code to create beneficial substitute molecules for patients with certain deficiencies.
Results
To unravel sequence mutations that destabilize amyloid fibrils yet simultaneously conserve native fold, we analyze the structural landscape of amyloid proteins and search for potential areas that could be exploited to weaken aggregation. Our tool, FibrilMutant, analyzes these regions and studies the effect of amino acid point mutations on nucleation and aggregation. This multiple objective approach impedes aggregation without stressing the cellular environment. We identified six main regions in amyloid proteins that contribute to structural stability and generated amino acid mutations to destabilize those regions. Full length fibrils were built from the mutated amyloid monomers and a dipolar-solvent model capturing the effect of dipole-dipole interactions between water and very large molecular systems to assess their aqueous stability was used to generate energy plots.
Conclusion
Our results are in agreement with experimental studies and suggest novel targeted single point mutations in the Amylin protein, potentially creating a better therapeutic agent than the currently administered Pramlintide drug for diabetes patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s12900-015-0034-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12900-015-0034-4
PMCID: PMC4428086  PMID: 25903685
Mutations; Amyloid; Fibrils; Amylin; Diabetes
8.  The observation of evolutionary interaction pattern pairs in membrane proteins 
Background
Over the last two decades, many approaches have been developed in bioinformatics that aim at one of the most promising, yet unsolved problems in modern life sciences - prediction of structural features of a protein. Such tasks addressed to transmembrane protein structures provide valuable knowledge about their three-dimensional structure. For this reason, the analysis of membrane proteins is essential in genomic and proteomic-wide investigations. Thus, many in-silico approaches have been utilized extensively to gain crucial advances in understanding membrane protein structures and functions.
Results
It turned out that amino acid covariation within interacting sequence parts, extracted from a evolutionary sequence record of α-helical membrane proteins, can be used for structure prediction. In a recent study we discussed the significance of short membrane sequence motifs widely present in nature that act as stabilizing ’building blocks’ during protein folding and in retaining the three-dimensional fold. In this work, we used motif data to define evolutionary interaction pattern pairs. These were obtained from different pattern alignments and were used to evaluate which coupling mechanisms the evolution provides. It can be shown that short interaction patterns of homologous sequence records are membrane protein family-specific signatures. These signatures can provide valuable information for structure prediction and protein classification. The results indicate a good agreement with recent studies.
Conclusions
Generally, it can be shown how the evolution contributes to realize covariation within discriminative interaction patterns to maintain structure and function. This points to their general importance for α-helical membrane protein structure formation and interaction mediation. In the process, no fundamentally energetic approaches of previous published works are considered. The low-cost rapid computational methods postulated in this work provides valuable information to classify unknown α-helical transmembrane proteins and to determine their structural similarity.
Electronic supplementary material
The online version of this article (doi:10.1186/s12900-015-0033-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12900-015-0033-5
PMCID: PMC4379700  PMID: 25887048
Membrane proteins; Motif; Evolutionary interaction pattern pair; EIPP; Structural similarity; Protein family affiliation
9.  Crystal structure of the DNA polymerase III β subunit (β-clamp) from the extremophile Deinococcus radiodurans 
Background
Deinococcus radiodurans is an extremely radiation and desiccation resistant bacterium which can tolerate radiation doses up to 5,000 Grays without losing viability. We are studying the role of DNA repair and replication proteins for this unusual phenotype by a structural biology approach. The DNA polymerase III β subunit (β-clamp) acts as a sliding clamp on DNA, promoting the binding and processivity of many DNA-acting proteins, and here we report the crystal structure of D. radiodurans β-clamp (Drβ-clamp) at 2.0 Å resolution.
Results
The sequence verification process revealed that at the time of the study the gene encoding Drβ-clamp was wrongly annotated in the genome database, encoding a protein of 393 instead of 362 amino acids. The short protein was successfully expressed, purified and used for crystallisation purposes in complex with Cy5-labeled DNA. The structure, which was obtained from blue crystals, shows a typical ring-shaped bacterial β-clamp formed of two monomers, each with three domains of identical topology, but with no visible DNA in electron density. A visualisation of the electrostatic surface potential reveals a highly negatively charged outer surface while the inner surface and the dimer forming interface have a more even charge distribution.
Conclusions
The structure of Drβ-clamp was determined to 2.0 Å resolution and shows an evenly distributed electrostatic surface charge on the DNA interacting side. We hypothesise that this charge distribution may facilitate efficient movement on encircled DNA and help ensure efficient DNA metabolism in D. radiodurans upon exposure to high doses of ionizing irradiation or desiccation.
doi:10.1186/s12900-015-0032-6
PMCID: PMC4350885  PMID: 25886944
DNA polymerase III β subunit; Deinococcus radiodurans; Radiation resistance
10.  High-resolution structure of a type IV pilin from the metal-reducing bacterium Shewanella oneidensis 
Background
Type IV pili are widely expressed among Gram-negative bacteria, where they are involved in biofilm formation, serve in the transfer of DNA, motility and in the bacterial attachment to various surfaces. Type IV pili in Shewanella oneidensis are also supposed to play an important role in extracellular electron transfer by the attachment to sediments containing electron acceptors and potentially forming conductive nanowires.
Results
The potential nanowire type IV pilin PilBac1 from S. oneidensis was characterized by a combination of complementary structural methods and the atomic structure was determined at a resolution of 1.67 Å by X-ray crystallography. PilBac1 consists of one long N-terminal α-helix packed against four antiparallel β-strands, thus revealing the core fold of type IV pilins. In the crystal, PilBac1 forms a parallel dimer with a sodium ion bound to one of the monomers. Interestingly, our PilBac1 crystal structure reveals two unusual features compared to other type IVa pilins: an unusual position of the disulfide bridge and a straight α-helical section, which usually exhibits a pronounced kink. This straight helix leads to a distinct packing in a filament model of PilBac1 based on an EM model of a Neisseria pilus.
Conclusions
In this study we have described the first structure of a pilin from Shewanella oneidensis. The structure possesses features of the common type IV pilin core, but also exhibits significant variations in the α-helical part and the D-region.
Electronic supplementary material
The online version of this article (doi:10.1186/s12900-015-0031-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s12900-015-0031-7
PMCID: PMC4376143  PMID: 25886849
Type IV pili; Nanowire; PilBac1; PilA; Shewanella oneidensis; X-Ray Crystallography; SAXS; SRCD
11.  BMC Structural Biology reviewer acknowledgment 2014 
doi:10.1186/s12900-015-0029-1
PMCID: PMC4330647
12.  Crystal structure and biophysical characterization of the nucleoside diphosphate kinase from Leishmania braziliensis 
Background
Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme that plays key roles in nucleotide recycling and homeostasis in trypanosomatids. It is also secreted by the intracellular parasite Leishmania to modulate the host response. These functions make NDK an attractive target for drug design and for studies aiming at a better understanding of the mechanisms mediating host-pathogen interactions.
Results
We report the crystal structure and biophysical characterization of the NDK from Leishmania braziliensis (LbNDK). The subunit consists of six α-helices along with a core of four β-strands arranged in a β2β3β1β4 antiparallel topology order. In contrast to the NDK from L. major, the LbNDK C-terminal extension is partially unfolded. SAXS data showed that LbNDK forms hexamers in solution in the pH range from 7.0 to 4.0, a hydrodynamic behavior conserved in most eukaryotic NDKs. However, DSF assays show that acidification and alkalization decrease the hexamer stability.
Conclusions
Our results support that LbNDK remains hexameric in pH conditions akin to that faced by this enzyme when secreted by Leishmania amastigotes in the parasitophorous vacuoles (pH 4.7 to 5.3). The unusual unfolded conformation of LbNDK C-terminus decreases the surface buried in the trimer interface exposing new regions that might be explored for the development of compounds designed to disturb enzyme oligomerization, which may impair the important nucleotide salvage pathway in these parasites.
doi:10.1186/s12900-015-0030-8
PMCID: PMC4322457  PMID: 25643978
Nucleoside diphosphate kinase; Leishmania braziliensis; Quaternary structure; Conformational stability
13.  Structural importance of the C-terminal region in pig aldo-keto reductase family 1 member C1 and their effects on enzymatic activity 
Background
Pig aldo-keto reductase family 1 member C1 (AKR1C1) belongs to AKR superfamily which catalyzes the NAD(P)H-dependent reduction of various substrates including steroid hormones. Previously we have reported two paralogous pig AKR1C1s, wild-type AKR1C1 (C-type) and C-terminal-truncated AKR1C1 (T-type). Also, the C-terminal region significantly contributes to the NADPH-dependent reductase activity for 5α-DHT reduction. Molecular modeling studies combined with kinetic experiments were performed to investigate structural and enzymatic differences between wild-type AKR1C1 C-type and T-type.
Results
The results of the enzyme kinetics revealed that Vmax and kcat values of the T-type were 2.9 and 1.6 folds higher than those of the C-type. Moreover, catalytic efficiency was also 1.9 fold higher in T-type compared to C-type. Since x-ray crystal structures of pig AKR1C1 were not available, three dimensional structures of the both types of the protein were predicted using homology modeling methodology and they were used for molecular dynamics simulations. The structural comparisons between C-type and T-type showed that 5α-DHT formed strong hydrogen bonds with catalytic residues such as Tyr55 and His117 in T-type. In particular, C3 ketone group of the substrate was close to Tyr55 and NADPH in T-type.
Conclusions
Our results showed that 5α-DHT binding in T-type was more favorable for catalytic reaction to facilitate hydride transfer from the cofactor, and were consistent with experimental results. We believe that our study provides valuable information to understand important role of C-terminal region that affects enzymatic properties for 5α-DHT, and further molecular mechanism for the enzyme kinetics of AKR1C1 proteins.
doi:10.1186/s12900-014-0028-7
PMCID: PMC4310174  PMID: 25583233
Aldo-keto reductase; Homology modeling; Molecular dynamic simulation; NADPH-dependent reduction; Steroid hormone
14.  A three dimensional visualisation approach to protein heavy-atom structure reconstruction 
Background
A commonly recurring problem in structural protein studies, is the determination of all heavy atom positions from the knowledge of the central α-carbon coordinates.
Results
We employ advances in virtual reality to address the problem. The outcome is a 3D visualisation based technique where all the heavy backbone and side chain atoms are treated on equal footing, in terms of the Cα coordinates. Each heavy atom is visualised on the surfaces of a different two-sphere, that is centered at another heavy backbone and side chain atoms. In particular, the rotamers are visible as clusters, that display a clear and strong dependence on the underlying backbone secondary structure.
Conclusions
We demonstrate that there is a clear interdependence between rotameric states and secondary structure. Our method easily detects those atoms in a crystallographic protein structure which are either outliers or have been likely misplaced, possibly due to radiation damage. Our approach forms a basis for the development of a new generation, visualization based side chain construction, validation and refinement tools. The heavy atom positions are identified in a manner which accounts for the secondary structure environment, leading to improved accuracy.
Electronic supplementary material
The online version of this article (doi:10.1186/s12900-014-0027-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12900-014-0027-8
PMCID: PMC4302604  PMID: 25551190
Side chain reconstruction; Cα trace problem; Rotamers; Protein visualisation
15.  Structural basis of RGD-hirudin binding to thrombin: Tyr3 and five C-terminal residues are crucial for inhibiting thrombin activity 
Background
Hirudin is an anti-coagulation protein produced by the salivary glands of the medicinal leech Hirudomedicinalis. It is a powerful and specific thrombin inhibitor. The novel recombinant hirudin, RGD-hirudin, which contains an RGD motif, competitively inhibits the binding of fibrinogen to GPIIb/IIIa on platelets, thus inhibiting platelet aggregation while maintaining its anticoagulant activity.
Results
Recombinant RGD-hirudin and six mutant variants (Y3A, S50A, Q53A, D55A, E57A and I59A), designed based on molecular simulations, were expressed in Pichia pastoris. The proteins were refolded and purified to homogeneity as monomers by gel filtration and anion exchange chromatography. The anti-thrombin activity of the six mutants and RGD-hirudin was tested. Further, we evaluated the binding of the mutant variants and RGD-hirudin to thrombin using BIAcore surface plasmon resonance analysis (SPR). Kinetics and affinity constants showed that the KD values of all six mutant proteins were higher than that of RGD-hirudin.
Conclusions
These findings contribute to a novel understanding of the interaction between RGD-hirudin and thrombin.
doi:10.1186/s12900-014-0026-9
PMCID: PMC4304120  PMID: 25526801
Recombinant RGD-hirudin; Thrombin; Molecular simulation; Surface plasmon resonance; Affinity constants
16.  In vitro histone lysine methylation by NSD1, NSD2/MMSET/WHSC1 and NSD3/WHSC1L 
Background
Histone lysine methylation has a pivotal role in regulating the chromatin. Histone modifiers, including histone methyl transferases (HMTases), have clear roles in human carcinogenesis but the extent of their functions and regulation are not well understood. The NSD family of HMTases comprised of three members (NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L) are oncogenes aberrantly expressed in several cancers, suggesting their potential to serve as novel therapeutic targets. However, the substrate specificity of the NSDs and the molecular mechanism of histones H3 and H4 recognition and methylation have not yet been established.
Results
Herein, we investigated the in vitro mechanisms of histones H3 and H4 recognition and modifications by the catalytic domain of NSD family members. In this study, we quantified in vitro mono-, di- and tri- methylations on H3K4, H3K9, H3K27, H3K36, H3K79, and H4K20 by the carboxyl terminal domain (CTD) of NSD1, NSD2 and NSD3, using histone as substrate. Next, we used a molecular modelling approach and docked 6-mer peptides H3K4 a.a. 1-7; H3K9 a.a. 5-11; H3K27 a.a. 23-29; H3K36 a.a. 32-38; H3K79 a.a. 75-81; H4K20 a.a. 16-22 with the catalytic domain of the NSDs to provide insight into lysine-marks recognition and methylation on histones H3 and H4.
Conclusions
Our data highlight the versatility of NSD1, NSD2, and NSD3 for recognizing and methylating several histone lysine marks on histones H3 and H4. Our work provides a basis to design selective and specific NSDs inhibitors. We discuss the relevance of our findings for the development of NSD inhibitors amenable for novel chemotherapies.
Electronic supplementary material
The online version of this article (doi:10.1186/s12900-014-0025-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12900-014-0025-x
PMCID: PMC4280037  PMID: 25494638
Epigenetic therapy of cancer; Histone lysine methyltransferase; NSD1; NSD2/MMSET/WHSC1; NSD3/WHSC1L; HMTase inhibitors
17.  Structural insights into Escherichia coli polymyxin B resistance protein D with X-ray crystallography and small-angle X-ray scattering 
Background
Polymyxin B resistance protein D (PmrD) plays a key role in the polymyxin B-resistance pathway, as it is the signaling protein that can act as a specific connecter between PmrA/PmrB and PhoP/PhoQ. We conducted structural analysis to characterize Escherichia coli (E. coli) PmrD, which exhibits different features compared with PmrD in other bacteria.
Results
The X-ray crystal structure of E. coli PmrD was determined at a 2.00 Å resolution, revealing novel information such as the unambiguous secondary structures of the protein and the presence of a disulfide bond. Furthermore, various assays such as native gel electrophoresis, surface plasmon resonance (SPR), size-exclusion chromatography, dynamic light scattering (DLS), and small-angle X-ray scattering (SAXS) measurements, were performed to elucidate the structural and functional role of the internal disulfide bond in E. coli PmrD.
Conclusions
The structural characteristics of E. coli PmrD were clearly identified via diverse techniques. The findings help explain the different protective mechanism of E. coli compared to other Gram-negative bacteria.
Electronic supplementary material
The online version of this article (doi:10.1186/s12900-014-0024-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s12900-014-0024-y
PMCID: PMC4263063  PMID: 25480013
PmrD; E. coli; SAXS; Crystal structure; Solution structure; Mutational study
18.  Structure and functional characterization of pyruvate decarboxylase from Gluconacetobacter diazotrophicus 
Background
Bacterial pyruvate decarboxylases (PDC) are rare. Their role in ethanol production and in bacterially mediated ethanologenic processes has, however, ensured a continued and growing interest. PDCs from Zymomonas mobilis (ZmPDC), Zymobacter palmae (ZpPDC) and Sarcina ventriculi (SvPDC) have been characterized and ZmPDC has been produced successfully in a range of heterologous hosts. PDCs from the Acetobacteraceae and their role in metabolism have not been characterized to the same extent. Examples include Gluconobacter oxydans (GoPDC), G. diazotrophicus (GdPDC) and Acetobacter pasteutrianus (ApPDC). All of these organisms are of commercial importance.
Results
This study reports the kinetic characterization and the crystal structure of a PDC from Gluconacetobacter diazotrophicus (GdPDC). Enzyme kinetic analysis indicates a high affinity for pyruvate (KM 0.06 mM at pH 5), high catalytic efficiencies (1.3 • 106 M−1•s−1 at pH 5), pHopt of 5.5 and Topt at 45°C. The enzyme is not thermostable (T½ of 18 minutes at 60°C) and the calculated number of bonds between monomers and dimers do not give clear indications for the relatively lower thermostability compared to other PDCs. The structure is highly similar to those described for Z. mobilis (ZmPDC) and A. pasteurianus PDC (ApPDC) with a rmsd value of 0.57 Å for Cα when comparing GdPDC to that of ApPDC. Indole-3-pyruvate does not serve as a substrate for the enzyme. Structural differences occur in two loci, involving the regions Thr341 to Thr352 and Asn499 to Asp503.
Conclusions
This is the first study of the PDC from G. diazotrophicus (PAL5) and lays the groundwork for future research into its role in this endosymbiont. The crystal structure of GdPDC indicates the enzyme to be evolutionarily closely related to homologues from Z. mobilis and A. pasteurianus and suggests strong selective pressure to keep the enzyme characteristics in a narrow range. The pH optimum together with reduced thermostability likely reflect the host organisms niche and conditions under which these properties have been naturally selected for. The lack of activity on indole-3-pyruvate excludes this decarboxylase as the enzyme responsible for indole acetic acid production in G. diazotrophicus.
Electronic supplementary material
The online version of this article (doi:10.1186/s12900-014-0021-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s12900-014-0021-1
PMCID: PMC4428508  PMID: 25369873
19.  A PDB-wide, evolution-based assessment of protein–protein interfaces 
Background
Thanks to the growth in sequence and structure databases, more than 50 million sequences are now available in UniProt and 100,000 structures in the PDB. Rich information about protein–protein interfaces can be obtained by a comprehensive study of protein contacts in the PDB, their sequence conservation and geometric features.
Results
An automated computational pipeline was developed to run our Evolutionary Protein–Protein Interface Classifier (EPPIC) software on the entire PDB and store the results in a relational database, currently containing > 800,000 interfaces. This allows the analysis of interface data on a PDB-wide scale. Two large benchmark datasets of biological interfaces and crystal contacts, each containing about 3000 entries, were automatically generated based on criteria thought to be strong indicators of interface type. The BioMany set of biological interfaces includes NMR dimers solved as crystal structures and interfaces that are preserved across diverse crystal forms, as catalogued by the Protein Common Interface Database (ProtCID) from Xu and Dunbrack. The second dataset, XtalMany, is derived from interfaces that would lead to infinite assemblies and are therefore crystal contacts. BioMany and XtalMany were used to benchmark the EPPIC approach. The performance of EPPIC was also compared to classifications from the Protein Interfaces, Surfaces, and Assemblies (PISA) program on a PDB-wide scale, finding that the two approaches give the same call in about 88% of PDB interfaces. By comparing our safest predictions to the PDB author annotations, we provide a lower-bound estimate of the error rate of biological unit annotations in the PDB. Additionally, we developed a PyMOL plugin for direct download and easy visualization of EPPIC interfaces for any PDB entry. Both the datasets and the PyMOL plugin are available at http://www.eppic-web.org/ewui/#downloads.
Conclusions
Our computational pipeline allows us to analyze protein–protein contacts and their sequence conservation across the entire PDB. Two new benchmark datasets are provided, which are over an order of magnitude larger than existing manually curated ones. These tools enable the comprehensive study of several aspects of protein–protein contacts in the PDB and represent a basis for future, even larger scale studies of protein–protein interactions.
doi:10.1186/s12900-014-0022-0
PMCID: PMC4274722  PMID: 25326082
Protein–protein interfaces; Biological interfaces; Crystal contacts; EPPIC; PISA; PDB
20.  Molecular dynamics simulations of the Nip7 proteins from the marine deep- and shallow-water Pyrococcus species 
Background
The identification of the mechanisms of adaptation of protein structures to extreme environmental conditions is a challenging task of structural biology. We performed molecular dynamics (MD) simulations of the Nip7 protein involved in RNA processing from the shallow-water (P. furiosus) and the deep-water (P. abyssi) marine hyperthermophylic archaea at different temperatures (300 and 373 K) and pressures (0.1, 50 and 100 MPa). The aim was to disclose similarities and differences between the deep- and shallow-sea protein models at different temperatures and pressures.
Results
The current results demonstrate that the 3D models of the two proteins at all the examined values of pressures and temperatures are compact, stable and similar to the known crystal structure of the P. abyssi Nip7. The structural deviations and fluctuations in the polypeptide chain during the MD simulations were the most pronounced in the loop regions, their magnitude being larger for the C-terminal domain in both proteins. A number of highly mobile segments the protein globule presumably involved in protein-protein interactions were identified. Regions of the polypeptide chain with significant difference in conformational dynamics between the deep- and shallow-water proteins were identified.
Conclusions
The results of our analysis demonstrated that in the examined ranges of temperatures and pressures, increase in temperature has a stronger effect on change in the dynamic properties of the protein globule than the increase in pressure. The conformational changes of both the deep- and shallow-sea protein models under increasing temperature and pressure are non-uniform. Our current results indicate that amino acid substitutions between shallow- and deep-water proteins only slightly affect overall stability of two proteins. Rather, they may affect the interactions of the Nip7 protein with its protein or RNA partners.
doi:10.1186/s12900-014-0023-z
PMCID: PMC4209456  PMID: 25315147
Molecular dynamics simulation; Nip7 protein; High pressure; Adaptation; Salt bridges
21.  Structural insight into the recognition of amino-acylated initiator tRNA by eIF5B in the 80S initiation complex 
Background
From bacteria to eukarya, the specific recognition of the amino-acylated initiator tRNA by the universally conserved translational GTPase eIF5B/IF2 is one of the most central interactions in the process of translation initiation. However, the molecular details, particularly also in the context of ribosomal initiation complexes, are only partially understood.
Results
A reinterpretation of the 6.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the eukaryal 80S initiation complex using the recently published crystal structure of eIF5B reveals that domain IV of eIF5B forms extensive interaction interfaces with the Met-tRNAi, which, in contrast to the previous model, directly involve the methionylated 3′ CCA-end of the acceptor stem. These contacts are mediated by a conserved surface area, which is homologous to the surface areas mediating the interactions between IF2 and fMet-tRNAfMet as well as between domain II of EF-Tu and amino-acylated elongator tRNAs.
Conclusions
The reported observations provide novel direct structural insight into the specific recognition of the methionylated acceptor stem by eIF5B domain IV and demonstrate its universality among eIF5B/IF2 orthologs in the three domains of life.
doi:10.1186/s12900-014-0020-2
PMCID: PMC4236685  PMID: 25350701
Ribosome; Translation initiation; Subunit joining; Initiator tRNA; eIF5B/IF2; Structure; Protein evolution
22.  A simple method for finding a protein’s ligand-binding pockets 
Background
This paper provides a simple and rapid method for a protein-clustering strategy. The basic idea implemented here is to use computational geometry methods to predict and characterize ligand-binding pockets of a given protein structure. In addition to geometrical characteristics of the protein structure, we consider some simple biochemical properties that help recognize the best candidates for pockets in a protein’s active site.
Results
Our results are shown to produce good agreement with known empirical results.
Conclusions
The method presented in this paper is a low-cost rapid computational method that could be used to classify proteins and other biomolecules, and furthermore could be useful in reducing the cost and time of drug discovery.
doi:10.1186/1472-6807-14-18
PMCID: PMC4112621  PMID: 25038637
Protein structure; Ligand-binding pockets; Computational methods
23.  Characterization of the SAM domain of the PKD-related protein ANKS6 and its interaction with ANKS3 
Background
Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder leading to end-stage renal failure in humans. In the PKD/Mhm(cy/+) rat model of ADPKD, the point mutation R823W in the sterile alpha motif (SAM) domain of the protein ANKS6 is responsible for disease. SAM domains are known protein-protein interaction domains, capable of binding each other to form polymers and heterodimers. Despite its physiological importance, little is known about the function of ANKS6 and how the R823W point mutation leads to PKD. Recent work has revealed that ANKS6 interacts with a related protein called ANKS3. Both ANKS6 and ANKS3 have a similar domain structure, with ankyrin repeats at the N-terminus and a SAM domain at the C-terminus.
Results
The SAM domain of ANKS3 is identified as a direct binding partner of the ANKS6 SAM domain. We find that ANKS3-SAM polymerizes and ANKS6-SAM can bind to one end of the polymer. We present crystal structures of both the ANKS3-SAM polymer and the ANKS3-SAM/ANKS6-SAM complex, revealing the molecular details of their association. We also learn how the R823W mutation disrupts ANKS6 function by dramatically destabilizing the SAM domain such that the interaction with ANKS3-SAM is lost.
Conclusions
ANKS3 is a direct interacting partner of ANKS6. By structurally and biochemically characterizing the interaction between the ANKS3 and ANKS6 SAM domains, our work provides a basis for future investigation of how the interaction between these proteins mediates kidney function.
doi:10.1186/1472-6807-14-17
PMCID: PMC4105859  PMID: 24998259
Polycystic kidney disease; Protein-protein interaction; Polymerization; Crystal structure
24.  High-resolution crystal structure of spin labelled (T21R1) azurin from Pseudomonas aeruginosa: a challenging structural benchmark for in silico spin labelling algorithms 
Background
EPR-based distance measurements between spin labels in proteins have become a valuable tool in structural biology. The direct translation of the experimental distances into structural information is however often impaired by the intrinsic flexibility of the spin labelled side chains. Different algorithms exist that predict the approximate conformation of the spin label either by using pre-computed rotamer libraries of the labelled side chain (rotamer approach) or by simply determining its accessible volume (accessible volume approach). Surprisingly, comparisons with many experimental distances have shown that both approaches deliver the same distance prediction accuracy of about 3 Å.
Results
Here, instead of comparing predicted and experimental distances, we test the ability of both approaches to predict the actual conformations of spin labels found in a new high-resolution crystal structure of spin labelled azurin (T21R1). Inside the crystal, the label is found in two very different environments which serve as a challenging test for the in silico approaches.
Conclusions
Our results illustrate why simple and more sophisticated programs lead to the same prediciton error. Thus, a more precise treatment of the complete environment of the label and also its interactions with the environment will be needed to increase the accuracy of in silico spin labelling algorithms.
doi:10.1186/1472-6807-14-16
PMCID: PMC4055355  PMID: 24884565
25.  Sequence analysis on the information of folding initiation segments in ferredoxin-like fold proteins 
Background
While some studies have shown that the 3D protein structures are more conservative than their amino acid sequences, other experimental studies have shown that even if two proteins share the same topology, they may have different folding pathways. There are many studies investigating this issue with molecular dynamics or Go-like model simulations, however, one should be able to obtain the same information by analyzing the proteins’ amino acid sequences, if the sequences contain all the information about the 3D structures. In this study, we use information about protein sequences to predict the location of their folding segments. We focus on proteins with a ferredoxin-like fold, which has a characteristic topology. Some of these proteins have different folding segments.
Results
Despite the simplicity of our methods, we are able to correctly determine the experimentally identified folding segments by predicting the location of the compact regions considered to play an important role in structural formation. We also apply our sequence analyses to some homologues of each protein and confirm that there are highly conserved folding segments despite the homologues’ sequence diversity. These homologues have similar folding segments even though the homology of two proteins’ sequences is not so high.
Conclusion
Our analyses have proven useful for investigating the common or different folding features of the proteins studied.
doi:10.1186/1472-6807-14-15
PMCID: PMC4055915  PMID: 24884463
Folding initiation segment prediction; Sequence analysis; Inter-residue average distance statistics; Evolutionarily conserved folding; Ribosomal protein S6; Procarboxypeptidase A2; U1A Spliceosomal protein; mt-Acylphosphatase

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