The macro domain as fusion tag for carrier‐driven crystallization

Obtaining well‐ordered crystals remains a significant challenge in protein X‐ray crystallography. Carrier‐driven crystallization can facilitate crystal formation and structure solution of difficult target proteins. We obtained crystals of the small and highly flexible SPX domain from the yeast vacuolar transporter chaperone 4 (Vtc4) when fused to a C‐terminal, non‐cleavable macro tag derived from human histone macroH2A1.1. Initial crystals diffracted to 3.3 Å resolution. Reductive protein methylation of the fusion protein yielded a new crystal form diffracting to 2.1 Å. The structures were solved by molecular replacement, using isolated macro domain structures as search models. Our findings suggest that macro domain tags can be employed in recombinant protein expression in E. coli, and in carrier‐driven crystallization.     

Protein Structure Prediction with a Combined Solvation Free Energy-Molecular Mechanics Force Field

Abstract

Models of protein structure are frequently used to determine the physical characteristics of a protein when the crystal structure is not available. We developed a procedure to optimize such models, by use of a combined solvation free energy and molecular mechanics force field. Appropriately chosen atomic solvation parameters were defined using the criterion that the resulting protein model should deviate least from the crystal structure upon a forty picosecond molecular dynamics simulation carried out using the combined force field. Several tests were performed to refine the set of atomic solvation parameters which best complement the molecular mechanics forces. Four sets of parameters from the literature were tested and an empirically optimized set is proposed. The parameters are defined on a well characterized small molecule (alanyl dipeptide) and on the highly refined crystal structure of rat trypsin, and then tested on a second highly refined crystal structure of α-lytic protease. The new set of atomic solvation parameters provides a significant improvement over molecular mechanics alone in energy minimization of protein structures. This combined force field also has advantages over the use of explicit solvent as it is possible to take solvent effects into account during energetic conformational searching when modeling a homologous protein structure from a known crystal structure.     

Protein purification and preliminary crystallographic analysis of human Lyn tyrosine kinase

Lyn is a member of the Src family of non-receptor protein kinase. As well as all members of the Src family, Lyn is thought to participate in signal transduction from cell surface receptors. The crystal structure of Lyn would have a better understanding of Lyn function in various cells. For the purpose of crystallization, C-terminal catalytic segment of human Lyn kinase conjugating hexahistidine purification tag (His-tag) was expressed in Sf21 insect cells. After first step purification utilizing His-tag, an anion-exchange chromatogram yielded four major peaks which had distinguishable phosphorylation manner as judged by Western blot analysis, Native-PAGE analysis and kinase activity measurements. The fractioned samples were separately examined for crystallization screening using a commercial available screening kit. The mono-phosphorylated protein was crystallized with a small rod-shaped and needle clusters. The higher phosphorylated samples corresponding to the other three fractions on the anion-exchange chromatogram were aggregated or precipitated under the above conditions. A crystal of the mono-phosphorylated sample was diffracted to 3.2 Å with synchrotron source at Photon Factory and a complete X-ray diffraction data set was collected. The coarse structure was solved by a molecular replacement method and further structural refinement is currently underway.     

Molecular replacement studies on crystal structure of DesBl-B2 Despentapeptide (B26-B30) insulin

Using the crystal structure of Despentapeptide (B26-B30) insulin (DPI) as the search model, the crystal structure of DesBl-B2 Despentapeptide (B26-B30) insulin (DesBl-2 DPI) has been studied by the molecular replacement method. There is one DesBl-2 DPI molecule in each crystallographic asymmetric unit. The cross rotation function search and the translation function search show apparent peaks and thus determine the orientation and position of DesBl-2 DPI molecule in the cell respectively. The subsequent three-dimensional structural rebuilding and refine-ment of DesBl-2 DPI molecule confirm the results by molecular replacement method.     

Crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum: evolutionary and mechanistic inferences for [3/4Fe-4S] ferredoxins.

The crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum has been solved by molecular replacement using data recorded with synchrotron radiation. The crystals were hexagonal prisms that showed a strong tendency to develop into long tubes. The hexagonal prisms diffracted to 2.1 A resolution at best, and a structural model for C. vinosum ferredoxin has been built with a final R of 19.2%. The N-terminal domain coordinates the two [4Fe-4S] clusters in a fold that is almost identical to that of other known ferredoxins. However, the structure has two unique features. One is a six-residue insertion between two ligands of one cluster forming a two-turn external loop; this short loop changes the conformation of the Cys 40 ligand compared to other ferredoxins and hampers the building of one NH...S H-bond to one of the inorganic sulfurs. The other remarkable structural element is a 3.5-turn alpha-helix at the C-terminus that covers one side of the same cluster and is linked to the cluster-binding domain by a six-residue external chain segment. The charge distribution is highly asymmetric over the molecule. The structure of C. vinosum ferredoxin strongly suggests divergent evolution for bacterial [3/4Fe-4S] ferredoxins from a common ancestral cluster-binding core. The unexpected slow intramolecular electron transfer rate between the clusters in C. vinosum ferredoxin, compared to other similar proteins, may be attributed to the unusual electronic properties of one of the clusters arising from localized changes in its vicinity rather than to a global structural rearrangement.     

Molecular replacement studies on crystal structure of DesB1-B2 Despentapeptide (B26-B30) insulin

Using the crystal structure of Despentapeptide (B26-B30) insulin (DPI as the search model, the crystal structure of DesB1-B2 Despentapeptide (B26-B30) insulin (DesB1-2 DPI) has been studied by the molecular replacement method. There is one DesB1-2 DPI molecule in each crystallographic asymmetric unit. The cross rotation function search and the translation function search show apparent peaks and thus determine the orientation and position of DesB1-2 DPI molecule in the cell respectively. The subsequent three-dimensional structural rebuilding and refinement of DesB1-2 DPI molecule confirm the results by molecular replacement method.     

Determination of protein oligomeric structure from small‐angle X‐ray scattering

Small‐angle X‐ray scattering (SAXS) is useful for determining the oligomeric states and quaternary structures of proteins in solution. The average molecular mass in solution can be calculated directly from a single SAXS curve collected on an arbitrary scale from a sample of unknown protein concentration without the need for beamline calibration or protein standards. The quaternary structure in solution can be deduced by comparing the experimental SAXS curve to theoretical curves calculated from proposed models of the oligomer. This approach is especially robust when the crystal structure of the target protein is known, and the candidate oligomer models are derived from the crystal lattice. When SAXS data are obtained at multiple protein concentrations, this analysis can provide insight into dynamic self‐association equilibria. Herein, we summarize the computational methods that are used to determine protein molecular mass and quaternary structure from SAXS data. These methods are organized into a workflow and demonstrated with four case studies using experimental SAXS data from the published literature.     

The effect of metal binding on the structure of annexin V and implications for membrane binding.

The structure of annexin V, crystallised in the presence of two calcium or barium ions for each protein molecule, was solved by molecular replacement to 0.24 nm resolution. The two metal ions are found in domains I and IV, i.e. on the same side of the channel that lies in the centre of the molecule. The structures of the barium and calcium form are extremely close, the only differences localised in the metal-binding sites that lie on the surface of the molecule. The occupancies of the metal ions, however, are lower for barium than for calcium, expressing the lower affinity of the protein for the former. The packing of the annexin molecules in the crystal asymmetric unit may represent a model for the calcium driven association of membrane-bound annexins that leads to membrane fusion.     

Crystal structure of the 16 heme cytochrome from Desulfovibrio gigas: a glycosylated protein in a sulphate-reducing bacterium

Sulphate-reducing bacteria have a wide variety of periplasmic cytochromes involved in electron transfer from the periplasm to the cytoplasm. HmcA is a high molecular mass cytochrome of 550 amino acid residues that harbours 16 c-type heme groups. We report the crystal structure of HmcA isolated from the periplasm of Desulfovibrio gigas. Crystals were grown using polyethylene glycol 8K and zinc acetate, and diffracted beyond 2.1 A resolution. A multiple-wavelength anomalous dispersion experiment at the iron absorption edge enabled us to obtain good-quality phases for structure solution and model building. DgHmcA has a V-shape architecture, already observed in HmcA isolated from Desulfovibrio vulgaris Hildenborough. The presence of an oligosaccharide molecule covalently bound to an Asn residue was observed in the electron density maps of DgHmcA and confirmed by mass spectrometry. Three modified monosaccharides appear at the highly hydrophobic vertex, possibly acting as an anchor of the protein to the cytoplasmic membrane.     

2.9 A resolution structure of the N-terminal domain of a variant surface glycoprotein from Trypanosoma brucei

The variant surface glycoprotein (VSG) of Trypanosoma brucei forms a coat on the surface of the parasite; by the expression of a series of antigenically distinct VSGs in the surface coat the parasite escapes the host immune response. The 2.9 A resolution crystal structure of the N-terminal domain of one variant, MITat 1.2, has been determined. The structure was solved using data collected from two crystal forms. Initially a partial model was built into an electron density map based on multiple isomorphous replacement phases and improved by phase combination methods. Subsequently this model was used to obtain the molecular replacement solution for a second crystal form, providing starting phases which were refined using 2-fold non-crystallographic symmetry averaging. The current model includes 362 residues and has been refined using X-PLOR to an R value of 0.22 for data between 7 and 2.9 A. The molecule is a dimer, approximately 100 A long, having an asymmetrical cross section with maximum dimensions of approximately 40 A x 60 A. Two long, approximately 70 A, alpha-helices from each monomer pack together to form, with several other helices, a core helix bundle that extends nearly the full length of the molecule. The "top" of the protein, which in the surface coat may be exposed to the external environment, is formed from the ends of the two long helices, a short three-stranded beta-sheet, and a strand having irregular conformation that packs above these secondary structure elements. Two conserved disulfide bridges are in this part of the molecule. Several elements of the MITat 1.2 sequence, which contribute to the formation of the helix bundle structure, have been identified. These elements can be found in the sequences of several different VSGs, suggesting that to some extent the VSG structure is conserved in those variants.     

Crystal structure of scaffolding protein CheW from thermoanaerobacter tengcongensis

The crystal structure of the scaffolding protein CheW from Thermoanaerobacter tengcongensis (TtCheW) is reported with a resolution at 2.2A using molecular replacement. Based on the crystal structure TmCheA P4-P5-TmCheW from Thermotoga maritime reported by others, we modeled the TmCheA P4-P5-TtCheW complex and predicted that TtCheW is involved in a hydrophobic interaction with CheA, similar to that for TmCheW. We also found that the conserved motif "NxxGxIxP" from CheW plays an important role in CheA binding. The coincidence of the reported mutation sites related to CheW-MCP binding, and the predicted protein interaction region within the TtCheW molecule, suggest that CheW-MCP binding sites lie in the groove-shaped area between TtCheW and the CheA P4 domain within the assembled model.     

Heterodimeric complex of RAR and RXR nuclear receptor ligand-binding domains: purification, crystallization, and preliminary X-ray diffraction analysis

Both the human retinoic acid receptor alpha (hRARalpha) and a constitutively active mutant (F318A) of the mouse retinoid X receptor alpha (mRXR alpha F318A) ligand-binding domains were separately overexpressed in Escherichia coli, copurified as a heterodimer in a two-step procedure, and cocrystallized with an RAR alpha-specific antagonist by using polyethylene glycol 10,000 as precipitant. The crystals grew in the hexagonal space group P6(1)22 displaying the unit cell parameters a = b = 116.6 A and c = 207.8 A. They diffracted X-ray to a limit of 2.2-A resolution. The asymmetric unit comprises one heterodimer and the crystal contains 60% solvent. The structure was determined by molecular replacement and is currently being refined.     

Three-dimensional structure of a mammalian purple acid phosphatase at 2.2 A resolution with a mu-(hydr)oxo bridged di-iron center.

The crystal structure of purple acid phosphatase from rat bone has been determined by molecular replacement and the structure has been refined to 2.2 A resolution to an R -factor of 21.3 % (R -free 26.5 %). The core of the enzyme consists of two seven-stranded mixed beta-sheets, with each sheet flanked by solvent-exposed alpha-helices on one side. The two sheets pack towards each other forming a beta-sandwich. The di-iron center, located at the bottom of the active-site pocket at one edge of the beta-sandwich, contains a mu-hydroxo or mu-oxo bridge and both metal ions are observed in an almost perfect octahedral coordination geometry. The electron density map indicates that a mu-(hydr)oxo bridge is found in the metal center and that at least one solvent molecule is located in the first coordination sphere of one of the metal ions. The crystallographic study of rat purple acid phosphatase reveals that the mammalian enzymes are very similar in overall structure to the plant enzymes in spite of only 18 % overall sequence identity. In particular, coordination and geometry of the iron cluster is preserved in both enzymes and comparison of the active-sites suggests a common mechanism for the mammalian and plant enzymes. However, significant differences are found in the architecture of the substrate binding pocket.     

Structure and function of the photosynthetic reaction center fromRhodobacter sphaeroides

The three-dimensional structure of the photosynthetic reaction center fromRhodobacter sphaeroides is described. The reaction center is a transmembrane protein that converts light into chemical energy. The protein has three subunits: L, M, and H. The mostly helical L and M subunits provide the scaffolding and the finely tuned environment in which the chromophores carry out electron transfer. The details of the protein-chromophore interactions are from studies of a trigonal crystal form that diffracted to 2.65-Å resolution. Functional studies of the multi-subunit complex by site-specific replacement of key amino acid residues are summarized in the context of the molecular structure.This work was supported in part by the U.S. Department of Energy, Office of Health and Environmental Research, under Contract No. W-31-109-ENG-38 and by Public Health Service Grant GM36598.     

The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity

Cas4 nucleases constitute a core family of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins, but little is known about their structure and activity. Here we report the crystal structure of the Cas4 protein Pcal_0546 from Pyrobaculum calidifontis, which revealed a monomeric protein with a RecB-like fold and one [2Fe-2S] cluster coordinated by four conserved Cys residues. Pcal_0546 exhibits metal-dependent 5′ to 3′ exonuclease activity against ssDNA substrates, whereas the Cas4 protein SSO1391 from Sulfolobus solfataricus can cleave ssDNA in both the 5′ to 3′ and 3′ to 5′ directions. The active site of Pcal_0546 contains a bound metal ion coordinated by the side chains of Asp123, Glu136, His146, and the main chain carbonyl of Ile137. Site-directed mutagenesis of Pcal_0546 and SSO1391 revealed that the residues of RecB motifs II, III and QhXXY are critical for nuclease activity, whereas mutations of the conserved Cys residues resulted in a loss of the iron-sulfur cluster, but had no effect on DNA cleavage. Our results revealed the biochemical diversity of Cas4 nucleases, which can have different oligomeric states, contain [4Fe-4S] or [2Fe-2S] clusters, and cleave single stranded DNA in different directions producing single-stranded DNA overhangs, which are potential intermediates for the synthesis of new CRISPR spacers.     

Computational approaches for protein function prediction: A combined strategy from multiple sequence alignment to molecular docking-based virtual screening

The functional characterization of proteins represents a daily challenge for biochemical, medical and computational sciences. Although finally proved on the bench, the function of a protein can be successfully predicted by computational approaches that drive the further experimental assays. Current methods for comparative modeling allow the construction of accurate 3D models for proteins of unknown structure, provided that a crystal structure of a homologous protein is available. Binding regions can be proposed by using binding site predictors, data inferred from homologous crystal structures, and data provided from a careful interpretation of the multiple sequence alignment of the investigated protein and its homologs. Once the location of a binding site has been proposed, chemical ligands that have a high likelihood of binding can be identified by using ligand docking and structure-based virtual screening of chemical libraries. Most docking algorithms allow building a list sorted by energy of the lowest energy docking configuration for each ligand of the library. In this review the state-of-the-art of computational approaches in 3D protein comparative modeling and in the study of protein–ligand interactions is provided. Furthermore a possible combined/concerted multistep strategy for protein function prediction, based on multiple sequence alignment, comparative modeling, binding region prediction, and structure-based virtual screening of chemical libraries, is described by using suitable examples. As practical examples, Abl-kinase molecular modeling studies, HPV-E6 protein multiple sequence alignment analysis, and some other model docking-based characterization reports are briefly described to highlight the importance of computational approaches in protein function prediction.     

A part of ice nucleation protein exhibits the ice-binding ability

We generated a recombinant 96-residue polypeptide corresponding to a sequence Tyr176-Gly273 of ice nucleation protein from Pseudomonas syringae (denoted INP96). INP96 exhibited an ability to shape an ice crystal, whose morphology is highly similar to the hexagonal-bipyramid generally identified for antifreeze protein. INP96 also showed a non-linear, concentration-dependent retardation of ice growth. Additionally, circular dichroism and NMR measurements suggested a local structural construction in INP96, which undergoes irreversible thermal denaturation. These data imply that a part of INP constructs a unique structure so as to interact with the ice crystal surfaces.     

Calculation of protein conformations by proton-proton distance constraints. A new efficient algorithm

We have developed a method to determine the three-dimensional structure of a protein molecule from such a set of distance constraints as can be determined by nuclear magnetic resonance studies. The currently popular methods for distance geometry based on the use of the metric matrix are applicable only to small systems. The method developed here is applicable to large molecules, such as proteins, with all atoms treated explicitly. This method works in the space of variable dihedral angles and determines a three-dimensional structure by minimization of a target function. We avoid difficulties hitherto inherent in this type of approach by two new devices: the use of variable target functions; and a method of rapid calculation of the gradient of the target functions. The method is applied to the determination of the structures of a small globular protein, bovine pancreatic trypsin inhibitor, from several artificial sets of distance constraints extracted from the X-ray crystal structure of this molecule. When a good set of constraints was available for both short- and long-range distances, the crystal structure was regenerated nearly exactly. When some ambiguities, such as those expected in experimental information, are allowed, the protein conformation can be determined up to a few local deformations. These ambiguities are mainly associated with the low resolving power of the short-range information.