Structural templates for modeling homodimers

Oligomeric proteins are more abundant in nature than monomeric proteins, and involved in all biological processes. In the absence of an experimental structure, their subunits can be modeled from their sequence like monomeric proteins, but reliable procedures to build the oligomeric assembly are scarce. Template‐based methods, which start from known protein structures, are commonly applied to model subunits. We present a method to model homodimers that relies on a structural alignment of the subunits, and test it on a set of 511 target structures recently released by the Protein Data Bank, taking as templates the earlier released structures of 3108 homodimeric proteins (H‐set), and 2691 monomeric proteins that form dimer‐like assemblies in crystals (M‐set). The structural alignment identifies a H‐set template for 97% of the targets, and in half of the cases, it yields a correct model of the dimer geometry and residue–residue contacts in the target. It also identifies a M‐set template for most of the targets, and some of the crystal dimers are very similar to the target homodimers. The procedure efficiently detects homology at low levels of sequence identities, and points to erroneous quaternary structures in the Protein Data Bank. The high coverage of the target set suggests that the content of the Protein Data Bank already approaches the structural diversity of protein assemblies in nature, and that template‐based methods should become the choice method for modeling oligomeric as well as monomeric proteins.     

A synergistic approach to protein crystallization: Combination of a fixed‐arm carrier with surface entropy reduction

Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier‐driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.     

Comparison of fluorescence and light scattering based methods to assess formation and stability of protein-protein complexes

Thermal shift methods such as differential scanning fluorimetry and differential static light scattering are widely used to identify stabilizing conditions for proteins that might promote crystallization. Here we report a comparison of the two methods when applied to optimization of buffer conditions for protein-protein complexes. Most of the protein complexes under study were amenable to analysis using these two techniques. Protein complexes behave towards thermal denaturation in a manner similar to single proteins, showing a more or less sharp transition consistent with a two-state model of unfolding. A comparison of the melting and aggregation temperatures for single components and the reconstituted complexes can provide additional evidence for complex formation and can be used to identify buffer conditions in which protein-protein complex formation is favored.     

Native fold and docking pose discrimination by the same residue‐based scoring function

Structure prediction and quality assessment are crucial steps in modeling native protein conformations. Statistical potentials are widely used in related algorithms, with different parametrizations typically developed for different contexts such as folding protein monomers or docking protein complexes. Here, we describe BACH‐SixthSense, a single residue‐based statistical potential that can be successfully employed in both contexts. BACH‐SixthSense shares the same approach as BACH, a knowledge‐based potential originally developed to score monomeric protein structures. A term that penalizes steric clashes as well as the distinction between polar and apolar sidechain‐sidechain contacts are crucial novel features of BACH‐SixthSense. The performance of BACH‐SixthSense in discriminating correctly the native structure among a competing set of decoys is significantly higher than other state‐of‐the‐art scoring functions, that were specifically trained for a single context, for both monomeric proteins (QMEAN, Rosetta, RF_CB_SRS_OD, benchmarked on CASP targets) and protein dimers (IRAD, Rosetta, PIE*PISA, HADDOCK, FireDock, benchmarked on 14 CAPRI targets). The performance of BACH‐SixthSense in recognizing near‐native docking poses within CAPRI decoy sets is good as well. Proteins 2015; 83:621–630. © 2015 Wiley Periodicals, Inc.     

Can the propensity of protein crystallization be increased by using systematic screening with metals?

Protein crystallization is one of the major bottlenecks in protein structure elucidation with new strategies being constantly developed to improve the chances of crystallization. Generally, well‐ordered epitopes possessing complementary surface and capable of producing stable inter‐protein interactions generate a regular three‐dimensional arrangement of protein molecules which eventually results in a crystal lattice. Metals, when used for crystallization, with their various coordination numbers and geometries, can generate such epitopes mediating protein oligomerization and/or establish crystal contacts. Some examples of metal‐mediated oligomerization and crystallization together with our experience on metal‐mediated crystallization of a putative rRNA methyltransferase from Sinorhizobium meliloti are presented. Analysis of crystal structures from protein data bank (PDB) using a non‐redundant data set with a 90% identity cutoff, reveals that around 67% of proteins contain at least one metal ion, with ～14% containing combination of metal ions. Interestingly, metal containing conditions in most commercially available and popular crystallization kits generally contain only a single metal ion, with combinations of metals only in a very few conditions. Based on the results presented in this review, it appears that the crystallization screens need expansion with systematic screening of metal ions that could be crucial for stabilizing the protein structure or for establishing crystal contact and thereby aiding protein crystallization.     

The dominating outer membrane protein of the hyperthermophilic Archaeum Ignicoccus hospitalis: a novel pore-forming complex

The membrane protein Imp1227 (Ignicoccus outer membrane protein; Imp1227) is the main protein constituent of the unique outer sheath of the hyperthermophilic, chemolithoautotrophic Archaeum Ignicoccus hospitalis. This outer sheath is the so far only known example for an asymmetric bilayer among the Archaea and is named 'outer membrane'. With its molecular mass of only 6.23 kDa, Imp1227 is found to be incorporated into the outer membrane in form of large, stable complexes. When separated by SDS-PAGE, they exhibit apparent masses of about 150, 50, 45 and 35 kDa. Dissociation into the monomeric form is achieved by treatment with SDS-containing solutions at temperatures at or above 113 degrees C. Electron micrographs of negatively stained samples confirm that isolated membranes are tightly packed with round complexes, about 7 nm in diameter, with a central, stain-filled 2 nm pore; a local two-dimensional crystalline arrangement in form of small patches can be detected by tomographic reconstruction. The comparison of the nucleotide and amino acid sequence of Imp1227 with public databases showed no reliable similarities with known proteins. Using secondary structure prediction and molecular modelling, an alpha-helical transmembrane domain is proposed; for the oligomer, a ring-shaped nonamer with a central 2 nm pore is a likely arrangement.     

A dissection of specific and non-specific protein-protein interfaces

We compare the geometric and physical-chemical properties of interfaces involved in specific and non-specific protein-protein interactions in crystal structures reported in the Protein Data Bank. Specific interactions are illustrated by 70 protein-protein complexes and by subunit contacts in 122 homodimeric proteins; non-specific interactions are illustrated by 188 pairs of monomeric proteins making crystal-packing contacts selected to bury more than 800 A2 of protein surface. A majority of these pairs have 2-fold symmetry and form "crystal dimers" that cannot be distinguished from real dimers on the basis of the interface size or symmetry. The chemical and amino acid compositions of the large crystal-packing interfaces resemble the protein solvent-accessible surface. These interfaces are less hydrophobic than in homodimers and contain much fewer fully buried atoms. We develop a residue propensity score and a hydrophobic interaction score to assess preferences seen in the chemical and amino acid compositions of the different types of interfaces, and we derive indexes to evaluate the atomic packing, which we find to be less compact at non-specific than at specific interfaces. We test the capacity of these parameters to identify homodimeric proteins in crystal structures, and show that a simple combination of the non-polar interface area and the fraction of buried interface atoms assigns the quaternary structure of 88% of the homodimers and 77% of the monomers in our data set correctly. These success rates increase to 93-95% when the residue propensity score of the interfaces is taken into consideration.     

Synthesis and functioning of the colicin E1 lysis protein: comparison with the colicin A lysis protein.

The colicin E1 lysis protein, CelA, was identified as a 3-kDa protein in induced cells of Escherichia coli K-12 carrying pColE1 by pulse-chase labeling with either [35S]cysteine or [3H]lysine. This 3-kDa protein was acylated, as shown by [2-3H]glycerol labeling, and seemed to correspond to the mature CelA protein. The rate of modification and processing of CelA was different from that observed for Cal, the colicin A lysis protein. In contrast to Cal, no intermediate form was detected for CelA, no signal peptide accumulated, and no modified precursor form was observed after globomycin treatment. Thus, the rate of synthesis would not be specific to lysis proteins. Solubilization in sodium dodecyl sulfate of the mature forms of both CelA and Cal varied similarly at the time of colicin release, indicating a change in lysis protein structure. This particular property would play a role in the mechanism of colicin export. The accumulation of the signal peptide seems to be a factor determining the toxicity of the lysis proteins since CelA provoked less cell damage than Cal. Quasi-lysis and killing due to CelA were higher in degP mutants than in wild-type cells. They were minimal in pldA mutants.     

Engineering a high-affinity scaffold for non-chromatographic protein purification via intein-mediated cleavage

While protein purification has long been dominated by standard chromatography, the relatively high cost and complex scale‐up have promoted the development of alternative non‐chromatographic separation methods. Here we developed a new non‐chromatographic affinity method for the purification of proteins expressed in Escherichia coli. The approach is to genetically fuse the target proteins with an affinity tag. Direct purification and recovery can be achieved using a thermo‐responsive elastin‐like protein (ELP) scaffold containing the capturing domain. Naturally occurring cohesin–dockerin pairs, which are high‐affinity protein complex responsible for the formation of cellulosome in anaerobic bacteria, were used as the model. By exploiting the highly specific interaction between the dockerin and cohesin domain from Clostridium thermocellum and the reversible aggregation property of ELP, highly purified and active dockerin‐tagged proteins, such as the endoglucanase CelA, chloramphenicol acetyl transferase (CAT), and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single thermal precipitation step with yields achieving over 90%. Incorporation of a self‐cleaving intein domain enabled rapid removal of the affinity tag from the target proteins, which was subsequently removed by another cycle of thermal precipitation. This method offers great flexibility as a wide range of affinity tags and ligands can be used. Biotechnol. Bioeng. 2012; 109: 2829–2835. © 2012 Wiley Periodicals, Inc.     

Analysis of Bacterial Immunoglobulin-Binding Proteins by X-Ray Crystallography

Protein crystallography offers a powerful means of analyzing the molecular mechanisms that underlie the action of bacterial immunoglobulin-binding proteins. Successful approaches used to date involve the isolation of individual IgG-binding domains from the immunoglobulin-binding protein under study and the crystallization of these on their own or in complex with Fc or Fab fragments. Two structures of complexes that have been determined to high resolution by protein crystallography are compared. A single IgG-binding domain from protein A (from Staphylococcus) binds to a human Fc fragment through formation of two α-helices, which bind in the cleft between the CH2 and the CH3 domains. Recognition is mediated by side chains on protein A which interact with conserved side chains on the surface of the antibody, ensuring binding to IgG molecules from different subclasses and species. A similar analysis of the complex of a single IgG-binding domain from protein G (from Streptococcus) with an Fab fragment from mouse IgG1 reveals that the same problem in molecular recognition is tackled in a different way. Protein G binds via an antiparallel alignment of β-strands from the IgG-binding domain and the CH1 domain in Fab: this main chain-main chain interaction is supported by a number of specific hydrogen bonds between the side chains in both proteins. By recognition of a high proportion of main-chain atoms, protein G minimizes the effects of IgG sequence variability in a way that is distinct from that adopted by protein A.     

Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments

Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three‐dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second‐generation anti‐EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although noncomplementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally‐determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well‐ordered surface features in our scFvs capable of forming versatile crystal contacts. Proteins 2014; 82:1884–1895. © 2014 Wiley Periodicals, Inc.     

The Dictyostelium discoideum spore germination-specific cellulase is organized into functional domains.

During Dictyostelium discoideum spore germination, degradation of the cellulose-containing spore wall is required to allow the amoeba to emerge. The CelA gene, which is transcribed and expressed exclusively during spore germination, codes for a 705-amino-acid protein that has cellulase activity [endo-(1,4)-beta-D-glucanase]. Amoebae transformed by a vector containing the CelA coding sequence or portions of it transcribed from a heterologous promoter expressed and secreted full-length or suitably truncated proteins during vegetative growth when, under normal conditions, these proteins are not made. The gene constructs divided the CelA protein into three domains: a 461-amino-acid N-terminal region that has significant similarity to those of other cellulases and that has been shown to be the catalytic domain; a contiguous 91-residue repeat containing the motif threonine-glutamic acid-threonine-proline, which is glycosylated; and, joined to the repeat, a C-terminal 153-amino-acid sequence that most probably defines a cellulose-binding domain.     

An approach to crystallizing proteins by metal-mediated synthetic symmetrization

Combining the concepts of synthetic symmetrization with the approach of engineering metal‐binding sites, we have developed a new crystallization methodology termed metal‐mediated synthetic symmetrization. In this method, pairs of histidine or cysteine mutations are introduced on the surface of target proteins, generating crystal lattice contacts or oligomeric assemblies upon coordination with metal. Metal‐mediated synthetic symmetrization greatly expands the packing and oligomeric assembly possibilities of target proteins, thereby increasing the chances of growing diffraction‐quality crystals. To demonstrate this method, we designed various T4 lysozyme (T4L) and maltose‐binding protein (MBP) mutants and cocrystallized them with one of three metal ions: copper (Cu²⁺), nickel (Ni²⁺), or zinc (Zn²⁺). The approach resulted in 16 new crystal structures—eight for T4L and eight for MBP—displaying a variety of oligomeric assemblies and packing modes, representing in total 13 new and distinct crystal forms for these proteins. We discuss the potential utility of the method for crystallizing target proteins of unknown structure by engineering in pairs of histidine or cysteine residues. As an alternate strategy, we propose that the varied crystallization‐prone forms of T4L or MBP engineered in this work could be used as crystallization chaperones, by fusing them genetically to target proteins of interest.     

Multiple precursor proteins bind individual Tat receptor complexes and are collectively transported

The thylakoid t win a rginine protein t ranslocation (Tat) system is thought to have a multivalent receptor complex with each cpTatC‐Hcf106 pair constituting a signal peptide‐binding unit. Conceptual models suggest that translocation of individual precursor proteins occurs upon assembly of a Tha4 oligomer with a precursor‐occupied cpTatC‐Hcf106. However, results reported here reveal that multiple precursor proteins bound to a single receptor complex can be transported together. Precursor proteins that contain one or two cysteine residues readily formed intermolecular disulphide bonds upon binding to the receptor complex, resulting in dimeric and tetrameric precursor proteins. Three lines of evidence indicate that all members of precursor oligomers were specifically bound to a receptor unit. Blue native–polyacrylamide gel electrophoresis analysis showed that oligomers were present on individual receptor complexes rather than bridging two or more receptor complexes. Upon energizing the membrane, the dimeric and tetrameric precursors were transported across the membrane with efficiencies comparable with that of monomeric precursors. These results imply a novel aspect of Tat systems, whereby multiple precursor‐binding sites can act in concert to transport an interlinked oligo‐precursor protein.     

Relative solvent accessible surface area predicts protein conformational changes upon binding

Protein interactions are often accompanied by significant changes in conformation. We have analyzed the relationships between protein structures and the conformational changes they undergo upon binding. Based upon this, we introduce a simple measure, the relative solvent accessible surface area, which can be used to predict the magnitude of binding-induced conformational changes from the structures of either monomeric proteins or bound subunits. Applying this to a large set of protein complexes suggests that large conformational changes upon binding are common. In addition, we observe considerable enrichment of intrinsically disordered sequences in proteins predicted to undergo large conformational changes. Finally, we demonstrate that the relative solvent accessible surface area of monomeric proteins can be used as a simple proxy for protein flexibility. This reveals a powerful connection between the flexibility of unbound proteins and their binding-induced conformational changes, consistent with the conformational selection model of molecular recognition.     

TAT‐MTS‐MCM fusion proteins reduce MMA levels and improve mitochondrial activity and liver function in MCM‐deficient cells

Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of the mitochondrial enzyme, methylmalonyl‐CoA mutase (MCM). The main treatments for MMA patients are dietary restriction of propiogenic amino acids and carnitine supplementation. Liver or combined liver/kidney transplantation has been used to treat those with the most severe clinical manifestations. Thus, therapies are necessary to help improve quality of life and prevent liver, renal and neurological complications. Previously, we successfully used the TAT‐MTS‐Protein approach for replacing a number of mitochondrial‐mutated proteins. In this targeted system, TAT, an 11 a.a peptide, which rapidly and efficiently can cross biological membranes, is fused to a mitochondrial targeting sequence (MTS), followed by the mitochondrial mature protein which sends the protein into the mitochondria. In the mitochondria, the TAT‐MTS is cleaved off and the native protein integrates into its natural complexes and is fully functional. In this study, we used heterologous MTSs of human, nuclear‐encoded mitochondrial proteins, to target the human MCM protein into the mitochondria. All fusion proteins reached the mitochondria and successfully underwent processing. Treatment of MMA patient fibroblasts with these fusion proteins restored mitochondrial activity such as ATP production, mitochondrial membrane potential and oxygen consumption, indicating the importance of mitochondrial function in this disease. Treatment with the fusion proteins enhanced cell viability and most importantly reduced MMA levels. Treatment also enhanced albumin and urea secretion in a CRISPR/Cas9‐engineered HepG2 MUT (‐/‐) liver cell line. Therefore, we suggest using this TAT‐MTS‐Protein approach for the treatment of MMA.     

Rapid automated detergent screening for the solubilization and purification of membrane proteins and complexes

Membrane proteins constitute about one third of proteins encoded by all genomes, but only a small percentage have their structures deposited in the Protein Data Bank. One bottleneck in the pipeline from expression to structure determination is the identification of detergents that maintain the protein in a soluble, stable, and active state. Here, we describe a small‐scale automated procedure to easily and rapidly screen detergents for the solubilization and purification of membrane proteins, to perform detergent exchange, or to identify conditions preserving protein interactions in complexes. Hundreds of conditions can be tested in a few hours to select detergents that keep proteins folded and nonaggregated, from single membrane preparations of cells overexpressing the protein(s) of interest. Thirty‐one prokaryotic, eukaryotic, and viral membrane proteins were analyzed by our small‐scale procedure to identify the best‐associated detergents. Examples of results obtained with a bitopic and multitopic membrane proteins and membrane protein complexes are presented in more detail. DDM, DM, DMNG, TritonX‐100, LAPAO, and Fos‐12 appeared effective for successful membrane solubilization and protein purification of most selected targets. Eukaryotic proteins are in general more difficult to extract and purify from Escherichia coli membranes than prokaryotic proteins. The protocol has been developed for His‐tagged proteins, but can readily be adapted to other affinity tags by adjusting the chromatography resin and the buffer composition.     

Production of small cysteine‐rich effector proteins in Escherichia coli for structural and functional studies

Although the lifestyles and infection strategies of plant pathogens are diverse, a prevailing feature is the use of an arsenal of secreted proteins, known as effectors, which aid in microbial infection. In the case of eukaryotic filamentous pathogens, such as fungi and oomycetes, effector proteins are typically dissimilar, at the protein sequence level, to known protein families and functional domains. Consequently, we currently have a limited understanding of how fungal and oomycete effectors promote disease. Protein biochemistry and structural biology are two methods that can contribute greatly to the understanding of protein function. Both techniques are dependent on obtaining proteins that are pure and functional, and generally require the use of heterologous recombinant protein expression systems. Here, we present a general scheme and methodology for the production and characterization of small cysteine‐rich (SCR) effectors utilizing Escherichia coli expression systems. Using this approach, we successfully produced cysteine‐rich effectors derived from the biotrophic fungal pathogen Melampsora lini and the necrotrophic fungal pathogen Parastagonospora nodorum. Access to functional recombinant proteins facilitated crystallization and functional experiments. These results are discussed in the context of a general workflow that may serve as a template for others interested in understanding the function of SCR effector(s) from their plant pathogen(s) of interest.     

The E. coli monothiol glutaredoxin GrxD forms homodimeric and heterodimeric FeS cluster containing complexes

Monothiol glutaredoxins (mono-Grx) represent a highly evolutionarily conserved class of proteins present in organisms ranging from prokaryotes to humans. Mono-Grxs have been implicated in iron sulfur (FeS) cluster biosynthesis as potential scaffold proteins and in iron homeostasis via an FeS-containing complex with Fra2p (homologue of E. coli BolA) in yeast and are linked to signal transduction in mammalian systems. However, the function of the mono-Grx in prokaryotes and the nature of an interaction with BolA-like proteins have not been established. Recent genome-wide screens for E. coli genetic interactions reported the synthetic lethality (combination of mutations leading to cell death; mutation of only one of these genes does not) of a grxD mutation when combined with strains defective in FeS cluster biosynthesis (isc operon) functions [Butland, G., et al. (2008) Nature Methods 5, 789-795]. These data connected the only E. coli mono-Grx, GrxD to a potential role in FeS cluster biosynthesis. We investigated GrxD to uncover the molecular basis of this synthetic lethality and observed that GrxD can form FeS-bound homodimeric and BolA containing heterodimeric complexes. These complexes display substantially different spectroscopic and functional properties, including the ability to act as scaffold proteins for intact FeS cluster transfer to the model [2Fe-2S] acceptor protein E. coli apo-ferredoxin (Fdx), with the homodimer being significantly more efficient. In this work, we functionally dissect the potential cellular roles of GrxD as a component of both homodimeric and heterodimeric complexes to ultimately uncover if either of these complexes performs functions linked to FeS cluster biosynthesis.