TIM Barrel Proteins Research Articles

Protection of catalytic cofactors by polypeptides as a driver for the emergence of primordial enzymes.

Enzymes catalyze the chemical reactions of life. For nearly half of known enzymes, catalysis requires the binding of small molecules known as cofactors. Polypeptide-cofactor complexes likely formed at a primordial stage and became starting points for the evolution of many efficient enzymes. Yet, evolution has no foresight so the driver for the primordial complex formation is unknown. Here, we use a resurrected ancestral TIM-barrel protein to identify one potential driver. Heme binding at a flexible region of the ancestral structure yields a peroxidation catalyst with enhanced efficiency when compared to free heme. This enhancement, however, does not arise from protein-mediated promotion of catalysis. Rather, it reflects protection of bound heme from common degradation processes and a resulting longer life time and higher effective concentration for the catalyst. Protection of catalytic cofactors by polypeptides emerges as a general mechanism to enhance catalysis and may have plausibly benefited primordial polypeptide-cofactor associations.

Loop dynamics and evolvability in the TIM-barrel proteins of histidine and tryptophan biosynthesis.

Recent years have witnessed increasing interest in the role of conformational dynamics in enzyme evolution and engineering. In this context, TIM-barrel proteins are important model systems, as the TIM-barrel is ubiquitous, highly evolvable, supports a diverse range of chemistry, and the active site is typically decorated by one or more mobile loops that play an important role in catalysis and/or regulation of these enzymes. Here, the TIM-barrel proteins of histidine and tryptophan biosynthesis (HisA, TrpF and PriA) stand out, as their active sites are decorated by up to three long mobile loops, that move independently of each other, and the conformations of which are critically important for catalysis. These motions appear to be ligand-gated and occur on sufficiently slow timescales (ms or slower) to be at least partially rate-limiting. In recent work, we combined conventional and enhanced molecular dynamics simulations with empirical valence bond simulations to investigate the conformational behavior of the catalytic loops in these enzymes, and the link between their dynamical behavior and substrate selectivity and catalysis. We have explored the role of loop dynamics in facilitating the bifunctionality of PriA towards substrates ProFAR and PRA (precursors for histidine and tryptophan biosynthesis, respectively), as well as during the real-time evolution of HisA to yield HisA-specific, TrpF-specific and bifunctional variants. Finally, the insights this provides emphasizes the potential of loop engineering as a powerful tool to manipulate the selectivity and substrate scope of the TIM-barrel scaffold, which has historically been one of the most important scaffolds for de novo enzyme design.

Rosetta's Predictive Ability for Low-Affinity Ligand Binding in Fragment-Based Drug Discovery.

Fragment-based drug discovery begins with the identification of small molecules with a molecular weight of usually less than 250 Da which weakly bind to the protein of interest. This technique is challenging for computational docking methods as binding is determined by only a few specific interactions. Inaccuracies in the energy function or slight deviations in the docking pose can lead to the prediction of incorrect binding or difficulties in ranking fragments in in silico screening. Here, we test RosettaLigand by docking a series of fragments to a cysteine-depleted variant of the TIM-barrel protein, HisF (UniProtKB Q9X0C6). We compare the computational results with experimental NMR spectroscopy screens. NMR spectroscopy gives details on binding affinities of individual ligands, which allows assessment of the ligand-ranking ability using RosettaLigand and also provides feedback on the location of the binding pocket, which serves as a reliable test of RosettaLigand's ability to identify plausible binding poses. From a library screen of 3456 fragments, we identified a set of 31 ligands with intrinsic affinities to HisF with dissociation constants as low as 400 μM. The same library of fragments was blindly screened in silico. RosettaLigand was able to rank binders before non-binders with an area under the curve of the receiver operating characteristics of 0.74. The docking poses observed for binders agreed with the binding pocket identified by NMR chemical shift perturbations for all fragments. Taken together, these results provide a baseline performance of RosettaLigand in a fragment-based drug discovery setting.

Complex Loop Dynamics Underpin Activity, Specificity, and Evolvability in the (βα)8 Barrel Enzymes of Histidine and Tryptophan Biosynthesis.

Enzymes are conformationally dynamic, and their dynamical properties play an important role in regulating their specificity and evolvability. In this context, substantial attention has been paid to the role of ligand-gated conformational changes in enzyme catalysis; however, such studies have focused on tremendously proficient enzymes such as triosephosphate isomerase and orotidine 5′-monophosphate decarboxylase, where the rapid (μs timescale) motion of a single loop dominates the transition between catalytically inactive and active conformations. In contrast, the (βα)8-barrels of tryptophan and histidine biosynthesis, such as the specialist isomerase enzymes HisA and TrpF, and the bifunctional isomerase PriA, are decorated by multiple long loops that undergo conformational transitions on the ms (or slower) timescale. Studying the interdependent motions of multiple slow loops, and their role in catalysis, poses a significant computational challenge. This work combines conventional and enhanced molecular dynamics simulations with empirical valence bond simulations to provide rich details of the conformational behavior of the catalytic loops in HisA, PriA, and TrpF, and the role of their plasticity in facilitating bifunctionality in PriA and evolved HisA variants. In addition, we demonstrate that, similar to other enzymes activated by ligand-gated conformational changes, loops 3 and 4 of HisA and PriA act as gripper loops, facilitating the isomerization of the large bulky substrate ProFAR, albeit now on much slower timescales. This hints at convergent evolution on these different (βα)8-barrel scaffolds. Finally, our work reemphasizes the potential of engineering loop dynamics as a tool to artificially manipulate the catalytic repertoire of TIM-barrel proteins.

Crystal structure of a phosphoribosyl anthranilate isomerase from the hyperthermophilic archaeon Thermococcus kodakaraensis.

A phosphoribosyl anthranilate isomerase, TkTrpF, from Thermococcus kodakaraensis was expressed in Escherichia coli and purified to homogeneity. TkTrpF was crystallized and its structure was determined by molecular replacement in two different space groups (C2 and P1) using data to 1.85 and 1.75 Å resolution, respectively. TkTrpF belongs to the class of TIM-barrel proteins. Structural comparison with other phosphoribosyl anthranilate isomerases (TrpFs) showed the highest structural similarity to Pyrococcus furiosus TrpF. Similarly to P. furiosus TrpF, TkTrpF is a monomer in solution, in contrast to other thermophilic enzymes, which exist as functional dimers. Although in space group P1 TkTrpF crystallizes with two molecules in the asymmetric unit, the interface is highly improbable in solution. Potential factors for the thermostability of TkTrpF were attributed to an increase in helical structure, an increased number of charged residues and an increase in the number of salt bridges.

De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy.

Despite efforts for over 25 years, de novo protein design has not succeeded in achieving the TIM-barrel fold. Here we describe the computational design of 4-fold symmetrical (β/α)8-barrels guided by geometrical and chemical principles. Experimental characterization of 33 designs revealed the importance of sidechain-backbone hydrogen bonding for defining the strand register between repeat units. The X-ray crystal structure of a designed thermostable 184-residue protein is nearly identical with the designed TIM-barrel model. PSI-BLAST searches do not identify sequence similarities to known TIM-barrel proteins, and sensitive profile-profile searches indicate that the design sequence is distant from other naturally occurring TIM-barrel superfamilies, suggesting that Nature has only sampled a subset of the sequence space available to the TIM-barrel fold. The ability to de novo design TIM-barrels opens new possibilities for custom-made enzymes.

Validation of the Conservation and Robustness of Folding Initiation Sites in Evolutionarily Related Proteins

Despite the recent progress in the protein folding field, how a given amino acid sequence folds into its unique structure is still unclear. According to KA Scott et. al., for Immunoglobulin and Fibronectin-like fold, which are evolutionarily unrelated but have the same topology, share a folding mechanism consisting of 4 key residues and their peripheral residues. However, there are also some cases that two evolutionarily related proteins have different folding mechanisms (for example, PDZ2 and PDZ3 whose sequence identity is nearly 30%). In this study, we aim to investigate whether such folding segments (i.e., folding initiation sites playing important roles for the folding from the denatured state to the transition state) really exist in evolutionarily related proteins by our sequence analysis methods and validate the results with the experimental phi values that provide a degree of the contribution for the structural formation in the transition state for each residue. Our sequence analysis methods are based on the inter-residue average distance statistics and already applied to several proteins, like leghemoglobin, FABP, azurin, and TIM-barrel proteins; they returned reasonable results with HD exchange experiments or phi-value analyses. According to the current study, for the Ferredoxin-like proteins we treat here, the predicted folding initiation sites have good agreements with experimental phi values, and the application to their evolutionarily related proteins returns a suggestion that the location of folding initiation sites are more conservative than sequence. In addition to these analyses, we also investigate the robustness of folding initiation sites by comparing the results of our analyses between naturally evolved proteins, which are under the pressure of nature, and artificially evolved proteins, which are not under the pressure of nature. The details will be reported in the conference.

Octarellin VI: Using Rosetta to Design a Putative Artificial (β/α)8 Protein

The computational protein design protocol Rosetta has been applied successfully to a wide variety of protein engineering problems. Here the aim was to test its ability to design de novo a protein adopting the TIM-barrel fold, whose formation requires about twice as many residues as in the largest proteins successfully designed de novo to date. The designed protein, Octarellin VI, contains 216 residues. Its amino acid composition is similar to that of natural TIM-barrel proteins. When produced and purified, it showed a far-UV circular dichroism spectrum characteristic of folded proteins, with α-helical and β-sheet secondary structure. Its stable tertiary structure was confirmed by both tryptophan fluorescence and circular dichroism in the near UV. It proved heat stable up to 70°C. Dynamic light scattering experiments revealed a unique population of particles averaging 4 nm in diameter, in good agreement with our model. Although these data suggest the successful creation of an artificial α/β protein of more than 200 amino acids, Octarellin VI shows an apparent noncooperative chemical unfolding and low solubility.

The role of Zn2+ on the structure and stability of murine adenosine deaminase.

Adenosine deaminase (ADA) is a key enzyme in purine metabolism and crucial for normal immune competence. It is a 40 kDa monomeric TIM-barrel protein containing a tightly bound Zn(2+), which is required for activity. In this study, we have investigated the role of Zn(2+) with respect to ADA structure and stability. After removing Zn(2+), the crystallographic structure of the protein remains highly ordered and similar to that of the holo protein with structural changes limited to regions capping the active site pocket. The stability of the protein, however, is decreased significantly in the absence of Zn(2+). Denaturation with urea shows the midpoint to be about 3.5 M for the apo enzyme, compared with 6.4 M for the holo enzyme. ADA contains four tryptophan residues distant from the Zn(2+) site. (19)F NMR studies in the presence and absence of Zn(2+) were carried out after incorporation of 6-(19)F-tryptophan. Chemical shift differences were observed for three of the four tryptophan residues, suggesting that, in contrast to the X-ray data, Zn(2+)-induced structural changes are propagated throughout the protein. Changes throughout the structure as suggested by the NMR data may explain the lower stability of the Zn(2+)-free protein. Real-time (19)F NMR spectroscopy measuring the loss of Zn(2+) showed that structural changes correlated with the loss of enzymatic activity.

TIM-Finder: A new method for identifying TIM-barrel proteins

BackgroundThe triosephosphate isomerase (TIM)-barrel fold occurs frequently in the proteomes of different organisms, and the known TIM-barrel proteins have been found to play diverse functional roles. To accelerate the exploration of the sequence-structure protein landscape in the TIM-barrel fold, a computational tool that allows sensitive detection of TIM-barrel proteins is required.ResultsTo develop a new TIM-barrel protein identification method in this work, we consider three descriptors: a sequence-alignment-based descriptor using PSI-BLAST e-values and bit scores, a descriptor based on secondary structure element alignment (SSEA), and a descriptor based on the occurrence of PROSITE functional motifs. With the assistance of Support Vector Machine (SVM), the three descriptors were combined to obtain a new method with improved performance, which we call TIM-Finder. When tested on the whole proteome of Bacillus subtilis, TIM-Finder is able to detect 194 TIM-barrel proteins at a 99% confidence level, outperforming the PSI-BLAST search as well as one existing fold recognition method.ConclusionsTIM-Finder can serve as a competitive tool for proteome-wide TIM-barrel protein identification. The TIM-Finder web server is freely accessible at http://202.112.170.199/TIM-Finder/.

Photoinduced, Family-Specific, Site-Selective Cleavage of TIM-Barrel Proteins

Nonenzymatic, chemical methods for the controlled cleavage of proteins at predictable sites in a site-specific manner are rare and of strong potential utility in clean, post-translational manipulation of protein structure for use in, for example, proteomics, sequencing, and tagged-protein production. Unprecedented photochemical, site-selective cleavage of a His-Trp (HW) motif in the GH1 family TIM-barrel proteins is observed upon exposure to 240-308 nm light to cleanly release N-terminal primary amide and C-terminal indolylenamide fragments. We also show that this photocleaveable motif can be transferred to fusion proteins for use in photoresponsive affinty purification. The presence of this motif in proteins found only in organisms that are not typically exposed to light raises the possibility of direct biological relevance for this new type of protein reaction.

Modeling and biochemical analysis of the activity of antibiofilm agent Dispersin B

Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix (PGA), which is a linear polymer of beta(1,6)-linked N-acetylglucosamine (GlcNAc) residues. Dispersin B (DspB), a soluble glycoside hydrolase produced by the periodontal pathogen Actinobacillus actinomycetemcomitans degrades PGA. The enzyme DspB is an alpha/beta TIM-barrel protein and belongs to family 20 glycosyl hydrolases members. The enzyme activity of DspB with regard to its substrate specificity towards beta(1,6)-linked GlcNAc polymers and its endo/exo character was investigated through ligand docking and the hydrolysis of synthetic oligosaccharides. Ligand docking analysis suggested that beta(1,6)-linked GlcNAc oligosaccharide bound to the active site better that beta(1,4)-linked GlcNAc oligosaccharide. Our combined results indicate that DspB is an exo-acting enzyme that hydrolyzes beta(1,6)-linked N-acetylglucosamine oligomers.

BCL::Align—Sequence alignment and fold recognition with a custom scoring function online

BCL::Align is a multiple sequence alignment tool that utilizes the dynamic programming method in combination with a customizable scoring function for sequence alignment and fold recognition. The scoring function is a weighted sum of the traditional PAM and BLOSUM scoring matrices, position-specific scoring matrices output by PSI-BLAST, secondary structure predicted by a variety of methods, chemical properties, and gap penalties. By adjusting the weights, the method can be tailored for fold recognition or sequence alignment tasks at different levels of sequence identity. A Monte Carlo algorithm was used to determine optimized weight sets for sequence alignment and fold recognition that most accurately reproduced the SABmark reference alignment test set. In an evaluation of sequence alignment performance, BCL::Align ranked best in alignment accuracy (Cline score of 22.90 for sequences in the Twilight Zone) when compared with Align-m, ClustalW, T-Coffee, and MUSCLE. ROC curve analysis indicates BCL::Align's ability to correctly recognize protein folds with over 80% accuracy. The flexibility of the program allows it to be optimized for specific classes of proteins (e.g. membrane proteins) or fold families (e.g. TIM-barrel proteins). BCL::Align is free for academic use and available online at http://www.meilerlab.org/.

Enzyme-Driven Speciation: Crystallizing Archaea via Lipid Capture

As the origin(s) of life on Earth remains an open question, detailed characteristics about the "last universal ancestor" (LUA) continue to be obscured. Here we provide arguments that strengthen the bacterial-like nature of the LUA. Our view attempts to recreate the evolution of archaeal lipids, the major components of the distinctive membrane that encapsulates these ancient prokaryotes. We show that (S)- 3-O-geranylgeranylglyceryl phosphate synthase (GGGPS), a TIM-barrel protein that performs the committed step in archaeal lipid synthesis, likely evolved from the duplication and fusion of a (betaalpha)4 half-barrel ancestor. By comparison to the well-characterized HisA and HisF TIM-barrel proteins, we propose a time line for the invention of this diagnostic archaeal biosynthetic pathway. After excluding the possibility of horizontal gene transfer, we conclude that the evolutionary history of GGGPS mirrors the emergence of Archaea from the LUA. We illustrate aspects of this "lipid capture" model that support its likelihood in recreating key evolutionary events and, as our hypothesis is built on a single initiating event, we suggest that the appearance of GGGPS represents an example of enzyme-driven speciation.

2P091 フラグメント化によるTIMバレルタンパク質のフォールディングコアの同定(蛋白質(物性(安定性、折れ畳みなど)),口頭発表,第45回日本生物物理学会年会)

2P091 フラグメント化によるTIMバレルタンパク質のフォールディングコアの同定(蛋白質(物性(安定性、折れ畳みなど)),口頭発表,第45回日本生物物理学会年会)

Exploring the environmental preference of weak interactions in (α/β)8 barrel proteins

The environmental preference for the occurrence of noncanonical hydrogen bonding and cation-pi interactions, in a data set containing 71 nonredundant (alpha/beta)(8) barrel proteins, with respect to amino acid type, secondary structure, solvent accessibility, and stabilizing residues has been performed. Our analysis reveals some important findings, which include (a) higher contribution of weak interactions mediated by main-chain atoms irrespective of the amino acids involved; (b) domination of the aromatic amino acids among interactions involving side-chain atoms; (c) involvement of strands as the principal secondary structural unit, accommodating cross strand ion pair interaction and clustering of aromatic amino acid residues; (d) significant contribution to weak interactions occur in the solvent exposed areas of the protein; (e) majority of the interactions involve long-range contacts; (f) the preference of Arg is higher than Lys to form cation-pi interaction; and (g) probability of theoretically predicted stabilizing amino acid residues involved in weak interaction is higher for polar amino acids such as Trp, Glu, and Gln. On the whole, the present study reveals that the weak interactions contribute to the global stability of (alpha/beta)(8) TIM-barrel proteins in an environment-specific manner, which can possibly be exploited for protein engineering applications.

Comparative model of EutB from coenzyme B12‐dependent ethanolamine ammonia‐lyase reveals a β8α8, TIM‐barrel fold and radical catalytic site structural features

The structure of the EutB protein from Salmonella typhimurium, which contains the active site of the coenzyme B12 (adenosylcobalamin)-dependent enzyme, ethanolamine ammonia-lyase, has been predicted by using structural proteomics techniques of comparative modelling. The 453-residue EutB protein displays no significant sequence identity with proteins of known structure. Therefore, secondary structure prediction and fold recognition algorithms were used to identify templates. Multiple three-dimensional template matching (threading) servers identified predominantly beta8alpha8, TIM-barrel proteins, and in particular, the large subunits of diol dehydratase (PDB: 1eex:A, 1dio:A) and glycerol dehydratase (PDB: 1mmf:A), as templates. Consistent with this identification, the dehydratases are, like ethanolamine ammonia-lyase, Class II coenzyme B12-dependent enzymes. Model building was performed by using MODELLER. Models were evaluated by using different programs, including PROCHECK and VERIFY3D. The results identify a beta8alpha8, TIM-barrel fold for EutB. The beta8alpha8, TIM-barrel fold is consistent with a central role of the alpha/beta-barrel structures in radical catalysis conducted by the coenzyme B12- and S-adenosylmethionine-dependent (radical SAM) enzyme superfamilies. The EutB model and multiple sequence alignment among ethanolamine ammonia-lyase, diol dehydratase, and glycerol dehydratase from different species reveal the following protein structural features: (1) a "cap" loop segment that closes the N-terminal region of the barrel, (2) a common cobalamin cofactor binding topography at the C-terminal region of the barrel, and (3) a beta-barrel-internal guanidinium group from EutB R160 that overlaps the position of the active-site potassium ion found in the dehydratases. R160 is proposed to have a role in substrate binding and radical catalysis.

The Crystal Structure of (S)-3-O-Geranylgeranylglyceryl Phosphate Synthase Reveals an Ancient Fold for an Ancient Enzyme

We report crystal structures of the citrate and sn-glycerol-1-phosphate (G1P) complexes of (S)-3-O-geranylgeranylglyceryl phosphate synthase from Archaeoglobus fulgidus (AfGGGPS) at 1.55 and 2.0 A resolution, respectively. AfGGGPS is an enzyme that performs the committed step in archaeal lipid biosynthesis, and it presents the first triose phosphate isomerase (TIM)-barrel structure with a prenyltransferase function. Our studies provide insight into the catalytic mechanism of AfGGGPS and demonstrate how it selects for the sn-G1P isomer. The replacement of "Helix 3" by a "strand" in AfGGGPS, a novel modification to the canonical TIM-barrel fold, suggests a model of enzyme adaptation that involves a "greasy slide" and a "swinging door." We propose functions for the homologous PcrB proteins, which are conserved in a subset of pathogenic bacteria, as either prenyltransferases or being involved in lipoteichoic acid biosynthesis. Sequence and structural comparisons lead us to postulate an early evolutionary history for AfGGGPS, which may highlight its role in the emergence of Archaea.

Rat liver betaine–homocysteine S-methyltransferase equilibrium unfolding: insights into intermediate structure through tryptophan substitutions

Equilibrium folding of rat liver BHMT (betaine-homocysteine methyltransferase), a TIM (triosephosphate isomerase)-barrel tetrameric protein, has been studied using urea as denaturant. A combination of activity measurements, tryptophan fluorescence, CD and sedimentation-velocity studies suggested a multiphasic process including two intermediates, a tetramer (I4) and a monomer (J). Analysis of denaturation curves for single- and six-tryptophan mutants indicated that the main changes leading to the tetrameric intermediate are related to alterations in the helix alpha4 of the barrel, as well as in the dimerization arm. Further dissociation to intermediate J included changes in the loop connecting the C-terminal alpha-helix of contact between dimers, disruption of helix alpha4, and initial alterations in helix alpha7 of the barrel, as well as in the dimerization arm. Evolution of the monomeric intermediate continued through additional perturbations in helix alpha7 of the barrel and the C-terminal loop. Our data highlight the essential role of the C-terminal helix in dimer-dimer binding through its contribution to the increased stability shown by BHMT as compared with other TIM barrel proteins. The results are discussed in the light of the high sequence conservation shown by betaine-homocysteine methyltransferases and the knowledge available for other TIM-barrel proteins.

SRide: a server for identifying stabilizing residues in proteins.

Residues expected to play key roles in the stabilization of proteins [stabilizing residues (SRs)] are selected by combining several methods based mainly on the interactions of a given residue with its spatial, rather than its sequential neighborhood and by considering the evolutionary conservation of the residues. A residue is selected as a stabilizing residue if it has high surrounding hydrophobicity, high long-range order, high conservation score and if it belongs to a stabilization center. The definition of all these parameters and the thresholds used to identify the SRs are discussed in detail. The algorithm for identifying SRs was originally developed for TIM-barrel proteins [M. M. Gromiha, G. Pujadas, C. Magyar, S. Selvaraj, and I. Simon (2004), Proteins, 55, 316–329] and is now generalized for all proteins of known 3D structure. SRs could be applied in protein engineering and homology modeling and could also help to explain certain folds with significant stability. The SRide server is located at .