In Advance

[ARTICLES] How well can the accuracy of comparative protein structure models be predicted?

Eramian, D., Eswar, N., Shen, M.-Y., Sali, A. — 2008-10-01

Comparative structure models are available for two orders of magnitude more protein sequences than are experimentally determined structures. These models, however, suffer from two limitations that experimentally determined structures do not: They frequently contain significant errors, and their accuracy cannot be readily assessed. We have addressed the latter limitation by developing a protocol optimized specifically for predicting the C root-mean-squared deviation (RMSD) and native overlap (NO3.5Å) errors of a model in the absence of its native structure. In contrast to most traditional assessment scores that merely predict one model is more accurate than others, this approach quantifies the error in an absolute sense, thus helping to determine whether or not the model is suitable for intended applications. The assessment relies on a model-specific scoring function constructed by a support vector machine. This regression optimizes the weights of up to nine features, including various sequence similarity measures and statistical potentials, extracted from a tailored training set of models unique to the model being assessed: If possible, we use similarly sized models with the same fold; otherwise, we use similarly sized models with the same secondary structure composition. This protocol predicts the RMSD and NO3.5Å errors for a diverse set of 580,317 comparative models of 6174 sequences with correlation coefficients (r) of 0.84 and 0.86, respectively, to the actual errors. This scoring function achieves the best correlation compared to 13 other tested assessment criteria that achieved correlations ranging from 0.35 to 0.71.

[FOR THE RECORD] The interaction of CK2{alpha} and CK2{beta}, the subunits of protein kinase CK2, requires CK2{beta} in a pre-formed conformation and is enthalpically driven

Raaf, J., Brunstein, E., Issinger, O.-G., Niefind, K. — 2008-09-29

The protein kinase CK2 (former name: "casein kinase 2") predominantly occurs as a heterotetrameric holoenzyme composed of two catalytic chains (CK2) and two non-catalytic subunits (CK2β). The CK2β subunits form a stable dimer to which the CK2 monomers are attached independently. In contrast to the cyclins in the case of the cyclin-dependent kinases CK2β is no on-switch of CK2; rather the formation of the CK2 holoenzyme is accompanied with an overall change of the enzyme's profile including a modulation of the substrate specifity, an increase of the thermostability and an allocation of docking sites for membranes and other proteins. In this study we used C-terminal deletion variants of human CK2 and CK2β that were enzymologically fully competent and in particular able to form a heterotetrameric holoenzyme. With differential scanning calorimetry (DSC) we confirmed the strong thermostabilization effect of CK2 on CK2β with an upshift of the CK2 melting temperature of more than nine degrees. Using isothermal titration calorimetry (ITC) we measured a dissociation constant of 12.6 nM. This high affinity between CK2 and CK2β is mainly caused by enthalpic rather than entropic contributions. Finally, we determined a crystal structure of the CK2β construct to 2.8 Å resolution and revealed by structural comparisons with the CK2 holoenzyme structure that the CK2β conformation is largely conserved upon association with CK2 whereas the latter undergoes significant structural adaptions of its backbone.

[ARTICLES] The I{kappa}B{alpha}/NF-{kappa}B complex has two hot-spots, one at either end of the interface

Bergqvist, S., Ghosh, G., Komives, E. A. — 2008-09-29

IB binds to and inhibits the transcriptional activity of NF-B family members via its ankyrin repeat (AR) domain. The binding affinity of IB with NF-B(p50/p65) heterodimers and NF-B(p65/65) homodimers is in the picomolar range and in the cell this results in long half-lives of the complexes. Direct binding experiments have been performed using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) on a series of truncations and mutations in order to understand what regions of the interface are most important for the tight binding affinity of this complex. We previously showed that interactions between residues 305-321 of NF-B(p65) with the first AR of IB are critical for the binding energy. Interactions in this region are responsible for more than 7 kcal/mol of the binding energy. Here we show equally drastic consequences for the binding energy occur upon truncation of even a few residues at the C-terminus of IB. Thus, the interface actually has two hot-spots, one at either end of the elongated and large surface of interaction. These results suggest a "squeeze" mechanism that leads to the extremely high affinity of the IB·NF-B complex through stabilization of the ankyrin repeat domain.

[UNSOLICITED REVIEW] Unconventional Serine Proteases: variations on the catalytic Ser/His/Asp triad configuration

Dogan Ekici, O., Paetzel, M., Dalbey, R. E — 2008-09-29

Serine proteases comprise nearly one-third of all known proteases identified to date and play crucial roles in a wide variety of cellular as well as extracellular functions including the process of blood clotting, protein digestion, cell signaling, inflammation and protein processing. Their hallmark is that they contain the so called "classical" catalytic Ser/His/Asp triad. Although the classical serine proteases are the most widespread in nature, there exist a variety of "non-classical" serine proteases where variations to the catalytic triad are observed. Such variations include the triads: Ser/His/Glu, Ser/His/His, Ser/Glu/Asp, and dyads: Ser/Lys and Ser/His. Other variations are seen with certain serine and threonine peptidases of the Ntn hydrolase superfamily that carry out catalysis with a single active site residue. This review discusses the structure and function of these novel serine proteases and threonine proteases, and how their catalytic machinery differs from the prototypic serine protease class.

[ARTICLES] Crystal Structures of Mycobacterium Tuberculosis S-Adenosyl-L-Homocysteine Hydrolase in Ternary Complex with Substrate and Inhibitors

2008-09-24

S-adenosylhomocysteine hydrolase (SAHH) is a ubiquitous enzyme that plays a central role in methylation-based processes by maintaining the intracellular balance between S-adenosylhomocysteine (SAH) and S-adenosylmethionine. We report the first prokaryotic crystal structure of SAHH from Mycobacterium tuberculosis (Mtb), in complex with adenosine (ADO) and nicotinamide adenine dinucleotide. Structures of complexes with three inhibitors are also reported: 3'-keto aristeromycin (ARI), 2-fluoroadenosine, and 3-deazaadenosine. The ARI complex is the first reported structure of SAHH complexed with this inhibitor, and confirms the oxidation of the 3' hydroxyl to a planar keto group, consistent with its prediction as a mechanism-based inhibitor. Enzyme activity and whole cell assays were carried out and the activities are consistent with the observed crystal structures. While most of the residues lining the ADO-binding pocket are identical between Mtb and human SAHH, less is known about the binding mode of the homocysteine (HCY) appendage of the full substrate. We report the 2.0 Å resolution structure of the complex of SAHH co-crystallized with SAH. The most striking change in the structure is that binding of HCY forces a rotation of His363 around the backbone to flip out of contact with the 5' hydroxyl of the ADO and opens access to a nearby channel that leads to the surface. This complex suggests that His363 acts as a switch that opens up to permit binding of substrate, then closes down after release of the cleaved HCY. Differences in the entrance to this access channel between human and Mtb SAHH are identified.

[PROTEIN STRUCTURE REPORTS] Solution structure of the extraterminal domain of the bromodomain-containing protein BRD4

2008-09-24

BRD4, which is a member of the BET (bromodomains and extraterminal) protein family, interacts preferentially with acetylated chromatin and possesses multiple cellular functions in meiosis, embryonic development, the cell cycle, and transcription. BRD4 and its family members contain two bromodomains known to bind acetylated lysine, and a conserved extraterminal (ET) domain whose function is unclear. Here we show the solution structure of the ET domain of mouse BRD4, which provides the first three-dimensional structure of an ET domain in the BET family. We determined the NMR structure of BRD4-ET with a root-mean-square deviation of 0.41 Å for the backbone atoms in the structured region of residues 608-676 on the basis of 1793 upper distance limits derived from NOE intensities measured in three-dimensional NOESY spectra. The structure of the BRD4-ET domain comprises three -helices and a characteristic loop region of irregular but well-defined structure. A DALI search revealed no close structural homologues in the current Protein Data Bank. The BRD4-ET structure has an acidic patch that forms a continuous ridge with a hydrophobic cleft, which may interact with other proteins and/or DNA.

[ARTICLES] Structural requirements for calmodulin binding to membrane-associated guanylate kinase homologs

Paarmann, I., Lye, M. F., Lavie, A., Konrad, M. — 2008-09-22

Effector molecules such as calmodulin modulate the interactions of membrane-associated guanylate kinase homologs (MAGUKs) and other scaffolding proteins of the membrane cytoskeleton by binding to the Src homology 3 (SH3) domain, the guanylate kinase (GK) domain, or the connecting HOOK region of MAGUKs. Using surface plasmon resonance, we studied the interaction of members of all four MAGUK subfamilies—synapse-associated protein 97 (SAP97), calcium/calmodulin-dependent serine protein kinase (CASK), membrane palmitoylated protein 2 (MPP2), and zona occludens (ZO) 1—and calmodulin to determine interaction affinities and localize the binding site. The SH3-GK domains of the proteins and derivatives thereof were expressed in E. coli and purified. In all four proteins, high-affinity calmodulin binding was identified. CASK was shown to contain a Ca²⁺-dependent calmodulin binding site within the HOOK region, overlapping with a protein 4.1 binding site. In ZO1, a Ca²⁺-dependent calmodulin binding site was detected within the GK domain. The equilibrium dissociation constants for MAGUK–calmodulin interaction were found to range from 50 nM to 180 nM. Sequence analyses suggest that binding sites for calmodulin have evolved independently in at least three subfamilies. For ZO1, pulldown of GST-calmodulin was shown to occur in a calcium-dependent manner; moreover, molecular modeling and sequence analyses predict conserved basic residues to be exposed on one side of a helix. Thus, calmodulin binding appears to be a common feature of MAGUKs, and Ca²⁺-activated calmodulin may serve as a general regulator to affect the interactions of MAGUKs and various components of the cytoskeleton.

[FOR THE RECORD] Computational models explain the oligosaccharide specificity of cyanovirin-N

Fujimoto, Y. K., Terbush, R. N., Patsalo, V., Green, D. F. — 2008-09-22

The prokaryotic lectin cyanovirin-N (CV-N) is a potent inhibitor of HIV envelope-mediated cell entry, and thus is a leading candidate among a new class of potential anti-HIV microbicides. The activity of CV-N is a result of interactions with the D1 arm of high-mannose oligosaccharides on the viral glycoprotein gp120. Here, we present computationally refined models of CV-N recognition of the di- and trisaccharides that represent the terminal three sugars of the D1 arm by each CV-N binding site. These models complement existing structural data, both from NMR spectroscopy and X-ray crystallography. When used with a molecular dynamics/continuum electrostatic (MD/PBSA) approach to compute binding free energies, these models explain the relative affinity of each site for the two saccharides. This work presents the first validation of the application of continuum electrostatic models to carbohydrate–protein association. Taken as a whole, the results both provide models of CV-N sugar recognition and demonstrate the utility of these computational methods for the study of carbohydrate-binding proteins.

[ARTICLES] Characterization of the steric defense of the HIV-1 gp41 N-trimer region

Eckert, D. M., Shi, Y., Kim, S., Welch, B. D., Kang, E., Poff, E. S., Kay, M. S. — 2008-09-18

During viral entry, HIV gp41 adopts a transient conformation called the 'prehairpin intermediate' in which a highly conserved therapeutic target, the N-trimer, is exposed. Despite extensive discovery efforts, potent and broadly neutralizing antibodies that target the N-trimer are elusive. We previously demonstrated the N-trimer is protected by a steric block that prevents large proteins, such as antibodies, from accessing it. Here we further characterize the steric block and identify its source. To study the N-trimer steric accessibility, we produced two sets of C-peptide inhibitors (a potent inhibitor targeting the N-trimer) fused to cargo proteins of increasing size facing either the virus or cell side of the prehairpin intermediate. Both bulky inhibitor sets show a steric block, but the effect is more pronounced with virus-side cargo. Additionally, both sets maintain their potencies in a modified entry assay that removes possible sources of target cell steric hindrance. These results implicate a viral source, likely gp120, as the primary component of the steric block. In addition, we studied the steric accessibility of the "pocket" region of the N-trimer, a highly attractive drug and vaccine target. We demonstrated a pocket-specific antibody, D5, is more potent as an scFv than as a full-length IgG, suggesting the N-trimer steric restriction extends to the pocket. This characterization of the steric block will facilitate the design of sterically restricted antigens that mimic the steric environment of the N-trimer in the prehairpin intermediate and are capable of inducing potent and broadly neutralizing antibodies that circumvent the N-trimer steric block.

[ARTICLES] Dynameomics: Large-Scale Assessment of Native Protein Flexibility

Benson, N., Daggett, V. — 2008-09-16

Structure is only the first step in understanding the interactions and functions of proteins. In this paper, we explore the flexibility of proteins across a broad database of over 250 solvated protein molecular dynamics simulations in water for an aggregate simulation time of ~6 ìs. These simulations are from our Dynameomics project, and these proteins represent approximately 75% of all known protein structures. We employ principal component analysis of the atomic coordinates over time to determine the primary axis and magnitude of the flexibility of each atom in a simulation. This technique gives us both a database of flexibility for many protein fold families and a compact visual representation of a particular protein's native-state conformational space, neither of which are available using experimental methods alone. These tools allow us to better understand the nature of protein motion and to describe its relationship to other structural and dynamical characteristics. In addition to reporting general properties of protein flexibility and detailing many dynamic motifs, we characterize the relationship between protein native-state flexibility and early events in thermal unfolding and show that flexibility predicts how a protein will begin to unfold. We provide evidence that fold families have conserved flexibility patterns and family members who deviate from the conserved patterns have very low sequence identity. Finally, we examine novel aspects of highly inflexible loops that are as important to structural integrity as conventional secondary structure. These loops, which are difficult if not impossible to locate without dynamic data, may constitute new structural motifs.

[ARTICLES] A double-headed cathepsin B inhibitor devoid of warhead

Schenker, P., Alfarano, P., Kolb, P., Caflisch, A., Baici, A. — 2008-09-16

Most synthetic inhibitors of peptidases have been targeted to the active site for inhibiting catalysis through reversible competition with the substrate or by covalent modification of catalytic groups. Cathepsin B is unique among the cysteine peptidase for the presence of a flexible segment, known as the occluding loop, which can block the primed subsites of the substrate binding cleft. With the occluding loop in the open conformation cathepsin B acts as an endopeptidase and as an exopeptidase when the loop is closed. We have targeted the occluding loop of human cathepsin B at its surface, outside the catalytic center, using a high-throughput docking procedure. The aim was to identify inhibitors that would interact with the occluding loop thereby modulating enzyme activity without the help of chemical warheads against catalytic residues. From a large library of compounds, the in silico approach identified [2-[2-(2,4-dioxo-1,3-thiazolidin-3-yl)ethylamino]-2-oxoethyl] 2-(furan-2-carbonylamino)acetate, which fulfils the working hypothesis. This molecule possesses two distinct binding moieties and behaves as a reversible, double-headed competitive inhibitor of cathepsin B by excluding synthetic and protein substrates from the active center. The kinetic mechanism of inhibition suggests that the occluding loop is stabilized in its closed conformation, mainly by hydrogen bonds with the inhibitor, thus decreasing endoproteolytic activity of the enzyme. Furthermore, the dioxothiazolidine head of the compound sterically hinders binding of the C-terminal residue of substrates resulting in inhibition of the exopeptidase activity of cathepsin B in a physiopathologically relevant pH range.

[ARTICLES] Physicochemical Changes in Phosphorylase Kinase Associated with Its Activation

Liu, W., Priddy, T. S., Carlson, G. M. — 2008-09-15

Phosphorylase kinase (PhK) regulates energy production through its Ca2+-dependent activation of glycogen phosphorylase in the cascade activation of glycogenolysis. The activity of PhK increases dramatically as the pH is raised from 6.8 to 8.2 (denoted as pH), but Ca2+-dependence is retained. Little is known about the structural changes associated with PhK's activation by pH and Ca2+, but activation by both mechanisms is mediated through regulatory subunits of the (β)4 PhK complex. In this study, changes in the structure of PhK induced by ¡üpH and Ca2+ were investigated using second derivative UV absorption, synchronous fluorescence, circular dichroism spectroscopy and zeta potential analyses. The joint effects of Ca2+ and pH on the physicochemical properties of PhK were found to be interdependent, with their effects showing a strong inflection point at ~ pH 7.6: above this pH, Ca2+ caused a large increase in the ratio of negative ellipticity at 222 and 208 nm; and below this pH, Ca2+ protected against a large decrease in zeta potential. Comparing the properties of the conformers of PhK present under the condition where it would be least active (pH 6.8 - Ca2+) versus that where it would be most active (pH 8.2 + Ca2+), the joint activation by pH and Ca2+ is characterized by a relatively large increase in the content of sheet structure, a decrease in interactions between helix and sheet structures, and a dramatically less negative electrostatic surface charge. A model is presented that accounts for the interdependent activating effects of pH and Ca2+ in terms of the overall physicochemical properties of the four PhK conformers described herein, and published data corroborating the transitions between these conformers are tabulated.

[ARTICLES] Proline 54 trans-cis isomerization is responsible for the kinetic partitioning at the last-step photocycle of photoactive yellow protein.

Lee, B.-C., Hoff, W. D. — 2008-09-15

Photoactive yellow protein (PYP), a blue-light photoreceptor for Ectothiorhodospira halophila, has provided a unique system for studying protein folding that is coupled with a photocycle. Upon receptor activation by blue light, PYP proceeds through a photocycle that includes a partially folded signaling state. The last-step photocycle is a thermal recovery reaction from the signaling state to the native state. Bi-exponential kinetics had been observed for the last-step photocycle, however the slow phase of the bi-exponential kinetics has not been extensively studied. Here we analyzed both fast and slow phase of the last-step photocycle in PYP. From the analysis of the denaturant dependence of the fast and slow phase, we found that the last-step photocycle proceeds through parallel channels of the folding pathway. The burial of solvent accessible area was responsible for the transition state of the fast phase, while structural rearrangement from the compact state to the native state was responsible for the transition state of the slow phase. The photocycle of PYP was linked to the thermodynamic cycle that includes both unfolding and refolding of the fast- and slow-phase intermediates. In order to test the hypothesis of proline-limited folding for the slow phase, we constructed two proline mutants P54A and P68A. We found that only a single phase of the last-step photocycle was observed in P54A. This suggests that there is a low energy barrier between trans to cis conformation in P54 in the light-induced state of PYP, and the resulting cis conformation of P54 generates a slow-phase kinetic trap during the photocycle-coupled folding pathway of PYP.

[ARTICLES] Novel affinity tag system using structurally defined antibody-tag interaction: Application to single-step protein purification

2008-09-11

Biologically important human proteins often require mammalian cell expression for structural studies, presenting technical and economical problems in the production/purification processes. We introduce novel affinity peptide tagging system that uses a low affinity anti-peptide monoclonal antibody. Concatenation of the short recognition sequence enabled the successful engineering of an 18-residue affinity tag with ideal solution binding kinetics, providing a low-cost purification means when combined with nondenaturing elution by water-miscible organic solvents. Three-dimensional information provides a firm structural basis for the antibody-peptide interaction, opening opportunities for further improvements/modifications.

[PROTEIN STRUCTURE REPORTS] Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri

2008-09-11

VirA, a secreted effector protein from Shigella sp., has been shown to be necessary for its virulence. It was also reported that VirA might be related to papain-like cysteine proteases and cleave -tubulin, thus facilitating intracellular spreading (Yoshida et al., Science 314, 985, 2006). We have now determined the crystal structure of VirA at 3.0 Å resolution. The shape of the molecule resembles the letter "V", with the residues in the N-terminal third of the 45 kDa molecule (some of which are disordered) forming one clearly identifiable domain, and the remainder of the molecule completing the V-like structure. The fold of VirA is unique and does not resemble that of any known protein, including papain, although its N-terminal domain is topologically similar to cysteine protease inhibitors such as stefin B. Analysis of the sequence conservation between VirA and its E. coli homologs EspG and EspG2 did not result in identification of any putative protease-like active site, leaving open a possibility that the biological function of VirA in Shigella virulence may not involve direct proteolytic activity.

[ARTICLES] Thioredoxin as a fusion-tag for carrier-driven crystallization

Corsini, L., Hothorn, M., Scheffzek, K., Sattler, M., Stier, G. — 2008-09-11

Structural investigations are frequently hindered by difficulties in obtaining diffracting crystals of the target protein. Here, we report the crystallization and structure solution of the UHM domain of splicing factor Puf60 fused to E. coli thioredoxin A. Both modules make extensive crystallographic contacts, contributing to a well-defined crystal lattice with clear electron density for both the thioredoxin and the Puf60-UHM module. We compare two short linker sequences between the two fusion domains, GSAM and GSPPM, for which only the GSAM-linked fusion protein yielded diffracting crystals. While specific interdomain contacts are not observed for both fusion proteins, NMR relaxation data in solution indicate reduced interdomain mobility between the Trx and Puf60-UHM modules. The GSPPM-linked fusion protein is significantly more flexible, albeit both linker sequences have the same number of degrees of torsional freedom. Our analysis provides a rationale for the crystallization of the GSAM-linked fusion protein and indicates that in this case, a four-residue linker between thioredoxin A and the fused target may represent the maximal length for crystallization purposes. Our data provide an experimental basis for the rational design of linker sequences in carrier-driven crystallization and identify thioredoxin A as a powerful fusion partner that can aid crystallization of difficult targets.

[ARTICLES] Conserved main-chain peptide distortions: a proposed role for Ile203 in catalysis by dihydrodipicolinate synthase.

2008-09-11

In recent years, dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) has received considerable attention from a mechanistic and structural view point. DHDPS catalyses the reaction of (S)-aspartate-β-semialdehyde with pyruvate, which is bound via a Schiff base to a conserved active-site lysine (Lys161 in the enzyme from Escherichia coli). To probe the mechanism of DHDPS, we have studied the inhibition of E. coli DHDPS by the substrate analogue, β-hydroxypyruvate. The Ki was determined to be 0.21 (±0.02) mM, similar to that of the allosteric inhibitor, (S)-lysine, and β-hydroxypyruvate was observed to cause time-dependent inhibition. The inhibitory reaction with β-hydroxypyruvate could be qualitatively followed by mass spectrometry, which showed initial non-covalent adduct formation, followed by the slow formation of the covalent adduct. It is unclear whether β-hydroxypyruvate plays a role in regulating the biosynthesis of meso-diaminopimelate and (S)-lysine in E. coli, although we note that it is present in vivo. The crystal structure of DHDPS complexed with β-hydroxypyruvate was solved. The active site clearly showed the presence of the inhibitor covalently bound to the Lys161. Interestingly, the hydroxyl group of β-hydroxypyruvate was hydrogen-bonded to the main-chain carbonyl of Ile203. This provides insight into the possible catalytic role played by this peptide unit, which has a highly strained torsion angle ( ~201°). A survey of the known DHDPS structures from other organisms shows this distortion to be a highly conserved feature of the DHDPS active site and we propose that this peptide unit plays a critical role in catalysis.

[FOR THE RECORD] Amino acid composition and protein dimension

Carugo, O. — 2008-09-09

There is indirect evidence that the amino acid composition of proteins depends on their dimension. The amino acid composition of a non redundant set of about 550,000 proteins was determined and it was observed that, in the range of 50-200 residues, the percentage of occurrence of most of the residue types significantly depends on protein dimension. This result should prove useful in analyzing protein sequences and genomics.

[ARTICLES] The Effects of Macromolecular Crowding on the Mechanical Stability of Protein Molecules

2008-09-09

Macromolecular crowding, a common phenomenon in the cellular environments, can significantly affect the thermodynamic and kinetic properties of proteins. A single-molecule method based on atomic force microscopy (AFM) was used to investigate the effects of macromolecular crowding on the forces required to unfold individual protein molecules. It was found that the mechanical stability of ubiquitin molecules was enhanced by macromolecular crowding from added dextran molecules. The average unfolding force increased from 210 pN in the absence of dextran to 234 pN in the presence of 300 g/l dextran at a pulling speed of 0.25 µm/s. A theoretical model, accounting for the effects of macromolecular crowding on the native and transition states of the protein molecule by applying the scaled particle theory, was used to quantitatively explain the crowding-induced increase in the unfolding force. The experimental results and interpretation presented could have wide implications for the many proteins that experience mechanical stresses and perform mechanical functions in the crowded environment of the cell.

[ARTICLES] Preventing Serpin Aggregation: The Molecular Mechanism of Citrate Action upon Antitrypsin Unfolding

2008-09-09

The aggregation of antitrypsin into polymers is one of the causes of neonatal hepatitis, cirrhosis and emphysema. A similar reaction resulting in disease can occur in other human serpins and collectively they are known as the serpinopathies. One possible therapeutic strategy involves inhibiting the conformational changes involved in antitrypsin aggregation. The citrate ion has previously been shown to prevent antitrypsin aggregation and maintain the protein in an active conformation; its mechanism of action however is unknown. Here we demonstrate that the citrate ion prevents the initial misfolding of the native state to a polymerogenic intermediate in a concentration dependent manner. Furthermore, we have solved the crystal structure of citrate bound to antitrypsin and show that a single citrate molecule binds in a pocket between the A and B β-sheets, a region known to be important in maintaining antitrypsin stability.

[ARTICLES] Refolding and simultaneous purification by three phase partitioning of recombinant proteins from inclusion bodies

2008-09-09

Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of three phase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding twelve different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies.

[ARTICLES] Structural and biochemical studies of TREX1 inhibition by metals. Identification of a new active histidine conserved in DEDDh exonucleases

Brucet, M., Querol-Audi, J., Bertlik, K., Lloberas, J., Fita, I., Celada, A. — 2008-09-09

TREX1 is the major exonuclease in mammalian cells, exhibiting the highest level of activity with a 3'->5' activity. This exonuclease is responsible in humans for Aicardi-Goutieres syndrome and for an autosomal dominant retinal vasculopathy with cerebral leukodystrophy. In addition, this enzyme is associated with systemic lupus erythematosus. TREX1 belongs to the exonuclease DEDDh family, whose members display low levels of sequence identity, while possessing a common fold and active site organization. For these exonucleases, a catalytic mechanism has been proposed that involves two divalent metal ions bound to the DEDD motif. Here we studied the interaction of TREX1 with the monovalent cations lithium and sodium. We demonstrate that these metals inhibit the exonucleolytic activity of TREX1, as measured by the classical gel method as well as by a new technique developed for monitoring the real-time exonuclease reaction. The X-ray structures of the enzyme in complex with these two cations and with a nucleotide, a product of the exonuclease reaction, were determined at 2.1 Å and 2.3 Å, respectively. A comparison with the structures of the active complexes (in the presence of magnesium or manganese) explains that the inhibition mechanism is caused by the non-catalytic metals competing with distinct affinities for the two metal-binding sites and inducing subtle rearrangements in active centers. Our analysis also reveals that a histidine residue (His124), highly conserved in the DEDDh family, is involved in the activity of TREX1, as confirmed by mutational studies. Our results shed further light on the mechanism of activity of the DEDEh family of exonucleases.

[ARTICLES] Three flavonoids targeting the {beta}-hydroxyacyl-acyl carrier protein dehydratase from Helicobacter pylori: crystal structure characterization with enzymatic inhibition assay

2008-09-09

Flavonoids are the major functional components of many herbal and insect preparations, and demonstrate varied pharmacological functions including antibacterial activity. Here by enzymatic assay and crystal structure analysis, we studied the inhibition of three flavonoids (quercetin, apigenin, and (S)-sakuranetin, Fig. 1) against the β-hydroxyacyl-acyl carrier protein dehydratase from Helicobacter pylori (HpFabZ). These three flavonoids are all competitive inhibitors against HpFabZ by either binding to the entrance of substrate tunnel B (binding model A) or plugging into the tunnel C near the catalytic residues (binding model B) mainly by hydrophobic interaction and hydrogen-bond pattern. Surrounded by hydrophobic residues of HpFabZ at both positions of models A and B, the methoxy group at C-7 of (S)-sakuranetin seems to play an important role for the inhibitor's binding to HpFabZ, partly responsible for the higher inhibitory activity of (S)-sakuranetin than those of quercetin and apigenin against HpFabZ (IC50 in µM: (S)-sakuranetin, 2.0±0.1; quercetin: 39.3±2.7; apigenin, 11.0±2.5). Our work is expected to supply useful information for understanding the potential antibacterial mechanism of flavonoids.

[ARTICLES] Fragment-HMM: A New Approach To Protein Structure Prediction

Li, S. C., Bu, D., Xu, J., Li, M. — 2008-08-25

We design a simple position-specific hidden Markov model to predict protein structure. Our new framework naturally repeats itself to converge to a final target, conglomerating fragment assembly, clustering, target selection, refinement, and consensus, all in one process. Our initial implementation of this theory converges to within 6 Angstrom of the native structures for 100% decoys on all 6 standard benchmark proteins used in ROSETTA which achieved only 14% to 94% for the same data. The qualities of the best decoys and the final decoys our theory converges to are also notably better.

[ARTICLES] Crystal Structure of the Human Receptor Activity-Modifying Protein 1 Extracellular Domain

2008-08-25

Receptor activity-modifying protein (RAMP) 1 forms a heterodimer with calcitonin receptor-like receptor (CRLR) and regulates its transport to the cell surface. The CRLR·RAMP1 heterodimer functions as a specific receptor for calcitonin gene-related peptide (CGRP). Here, we report the crystal structure of the human RAMP1 extracellular domain. The RAMP1 structure is a three-helix bundle that is stabilized by three disulfide bonds. The RAMP1 residues important for cell-surface expression of the CRLR·RAMP1 heterodimer are clustered to form a hydrophobic patch on the molecular surface. The hydrophobic patch is located near the tryptophan residue essential for binding of the CGRP antagonist, BIBN4096BS. These results suggest that the hydrophobic patch participates in the interaction with CRLR and the formation of the ligand-binding pocket when it forms the CRLR·RAMP1 heterodimer.

[ARTICLES] A Fast and Accurate Computational Approach to Protein Ionization

Spassov, V. Z, Yan, L. — 2008-08-19

We report a very fast and accurate physics-based method to calculate pH dependent electrostatic effects in protein molecules and to predict the pK values of individual sites of titration. In addition, a CHARMm based algorithm is included to construct and refine the spatial coordinates of all hydrogen atoms at a given pH. The present method combines electrostatic energy calculations based on the Generalized Born approximation with an iterative mobile clustering approach to calculate the equilibria of proton binding to multiple titration sites in protein molecules. The use of the GBIM (Generalized Born with Implicit Membrane) CHARMm module makes it possible to model not only water-soluble proteins but membrane proteins as well. The method includes a novel algorithm for preliminary refinement of hydrogen coordinates, including the determination of the optimal proton binding centers in ambiguous cases such as the carboxyl groups of acidic residues. Another difference from existing approaches is that instead of monopeptides, a set of relaxed pentapeptide structures are used as model compounds based on a recent accurate experimental estimation of the pKa values of natural acidic and basic residues in Ala-Ala-X-Ala-Ala structures. Tests on a set of 24 proteins demonstrate the high accuracy of the method. On average, the rmsd between predicted and experimental pK values is close to 0.5 pK units on this data set, and the accuracy is achieved at a very low computational cost. The pH-dependent assignment of hydrogen atoms also shows very good agreement with protonation states and hydrogen-bond network observed in neutron-diffraction structures. The protonation states and hydrogen positions are predicted correctly for 94% of all 849 residues in a test set of five structures, including 100% of tautomeric states of histidines and 82% of Asn and Gln amide groups. There is only one adjustable parameter in the method: the value of intra-molecular dielectric constant. All other parameters are kept at their standard force field values. The method is implemented as a computational protocol in Accelrys Discovery Studio 2.0 and provides a fast and easy way to study the effect of pH on many important mechanisms such as enzyme catalysis, ligand binding, protein-protein interactions, and protein stability.

[ARTICLES] Atomic Resolution Structures of Escherichia coli and Bacillus anthracis Malate Synthase A: Comparison with Isoform G and Implications for Structure Based Drug Discovery

Lohman, J. R., Olson, A. C., Remington, S. J. — 2008-08-19

Enzymes of the glyoxylate shunt are important for the virulence of pathogenic organisms such as Mycobacterium tuberculosis and Candida albicans. Two isoforms have been identified for malate synthase, the second enzyme in the pathway. Isoform A, found in fungi and plants, comprises ~530 residues, whereas isoform G, found only in bacteria, is larger by ~200 residues. Crystal structures of malate synthase isoform G from Escherichia coli and Mycobacterium tuberculosis were previously determined at moderate resolution. Here we describe crystal structures of Escherichia coli malate synthase A (MSA) in the apo form (1.04 Å resolution) and in complex with acetylcoenzyme A and a competitive inhibitor, possibly pyruvate or oxalate (1.40 Å resolution). In addition, a crystal structure for Bacillus anthracis MSA at 1.70 Å resolution is reported. The increase in size between isoforms A and G can be attributed primarily to an inserted /β domain that may have regulatory function. Upon binding of inhibitor or substrate, several active site loops in MSA undergo large conformational changes. However, in the substrate bound form, the active sites of isoforms A and G from Escherichia coli are nearly identical. Considering that inhibitors bind with very similar affinities to both isoforms, MSA is as an excellent platform for high-resolution structural studies and drug discovery efforts.

[ARTICLES] IDENTIFICATION OF SERINE 205 AS A SITE OF PHOSPHORYLATION ON PAX3 IN PROLIFERATING BUT NOT DIFFERENTIATING PRIMARY MYOBLASTS

Miller, P. J, Dietz, K. N, Hollenbach, A. D — 2008-08-15

Pax3, a member of the paired class homeodomain family of transcription factors, is essential for early skeletal muscle development. Previously, others and we have shown that the stability of Pax3 is regulated on a post-translational level. Evidence in the literature and from our lab suggests that phosphorylation, a common form of regulation, may play a role. However, at present, the sites of Pax3 phosphorylation are not known. We demonstrate here the first evidence that Pax3 exists as a phosphoprotein in proliferating mouse primary myoblasts. Using an in vitro kinase assay, deletion, and point mutant analysis, we conclusively identify Ser205 as a site of phosphorylation. The phosphorylation of Ser205 on endogenously expressed Pax3 was confirmed in vivo using antibodies specific for phosphorylation at Ser205. Finally, we demonstrate for the first time that the phosphorylation status of endogenous Pax3 changes rapidly upon the induction of myogenic differentiation. The presence of phosphorylation in a region of Pax3 important for mediating protein-protein interactions and the fact that phosphorylation is lost upon induction of differentiation allows for speculation on the biological relevance of phosphorylation.

[ARTICLES] Activation Profiles of Human Kallikrein-related Peptidases by Proteases of the Thrombostasis Axis

2008-08-12

The human kallikrein-related peptidases (KLKs) comprise fifteen members (KLK1-15) and are the single largest family of serine proteases. The KLKs are utilized, or proposed, as clinically important biomarkers and therapeutic targets of interest in cancer and neurodegenerative disease. All KLKs appear to be secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their amino-terminal pro-peptide. This processing is a key step in the regulation of KLK function. Much recent work has been devoted to elucidating the potential for activation cascades between members of the KLK family, with physiologically relevant KLK regulatory cascades now described in skin desquamation and semen liquefaction. Despite this expanding knowledge of KLK regulation, details regarding the potential for functional intersection of KLKs with other regulatory proteases are essentially unknown. To elucidate such interaction potential, we have characterized the ability of proteases associated with thrombostasis to hydrolyze the pro-peptide sequences of the KLK family using a previously described pro-KLK fusion protein system. A subset of positive hydrolysis results were subsequently quantified with proteolytic assays using intact recombinant pro-KLK proteins. Pro-KLK6 and 14 can be activated by both plasmin and uPA, with plasmin being the best activator of pro-KLK6 identified to date. Pro-KLK11 and 12 can be activated by a broad-spectrum of thrombostasis proteases, with thrombin exhibiting a high-degree of selectivity for pro-KLK12. The results show that proteases of the thrombostasis family can efficiently activate specific pro-KLKs, demonstrating the potential for important regulatory interactions between these two major protease families.

[ACCELERATED COMMUNICATIONS] An electrostatic network and long-range regulation of Src kinases

Ozkirimli, E., Yadav, S. S., Miller, T., Post, C. B. — 2008-08-07

The regulatory mechanism of Src tyrosine kinases includes conformational activation by a change in the catalytic domain tertiary structure and in domain domain contacts between the catalytic domain and the SH2/SH3 regulatory domains. The kinase is activated when tyrosine phosphorylation occurs on the activation loop but without phosphorylation of the C-terminal tail. Activation also occurs by allostery when contacts between the catalytic domain (CD) and the regulatory SH3 and SH2 domains are released as a result of exogenous protein binding. The aim of this work is to examine the proposed role of an electrostatic network in the conformational transition and to elucidate the molecular mechanism for long-range, allosteric conformational activation by a combination of experimental enzyme kinetics and non-equilibrium molecular dynamics simulations. A salt dependence of the induction phase is observed in kinetic assays and supports the role of an electrostatic network in the transition. In addition, simulations provide evidence that allosteric activation involves a concerted motion coupling highly conserved residues, and spanning several nm from the catalytic site to the regulatory domain interface for communication between CD and the regulatory domains.

[ARTICLES] Electrostatic Contributions Drive the Interaction Between Staphylococcus aureus Protein Efb-C and its Complement Target C3d

Haspel, N., Ricklin, D., Geisbrecht, B. V, Kavraki, L. E., Lambris, J. D. — 2008-08-07

The C3-inhibitory domain of Staphylococcus aureus extracellular fibrinogen-binding protein (Efb-C) defines a novel three-helix bundle motif that regulates complement activation. Previous crystallographic studies of Efb-C bound to its cognate sub-domain of human C3 (C3d) identified Arg-131 and Asn-138 of Efb-C as key residues for its activity. In order to characterize more completely the physical and chemical driving forces behind this important interaction, we employed in this study a combination of structural, biophysical, and computational methods to analyze the interaction of C3d with Efb-C and the single point mutants R131A and N138A. Our results show that while these mutations do not drastically affect the structure of the Efb-C/C3d recognition complex, they have significant adverse effects on both the thermodynamic kinetic profiles of the resulting complexes. We also characterized other key interactions along the Efb-C/C3d binding interface and found an intricate network of salt bridges and hydrogen bonds that anchor Efb-C to C3d, resulting in its potent complement inhibitory properties.

[FOR THE RECORD] Assignment-free solution NMR method reveals CesT as an unswapped homodimer

Rumpel, S., Lakshmi, R., Becker, S., Zweckstetter, M. — 2008-08-07

The X-ray structure of the homodimeric chaperone CesT is the only structure among the type three secretion system (TTSS) chaperones which shows a domain swap. This swap has potential importance for the mechanism of effector translocation through a TTSS. Here we present two NMR strategies exploiting preexisting structural models and residual dipolar couplings (RDCs), which reveal the unswapped 35.4 kDa dimer to be present in solution. Particularly efficient is the discrimination of a swapped and unswapped structural state performed simultaneously to automatic backbone assignment using only HN-RDCs and carbonyl backbone chemical shifts. This direct approach may prove to be generally useful to rapidly differentiate two structural models.

[ARTICLES] The solution structure of the C-terminal domain of NfeD reveals a novel membrane-anchored OB-fold.

2008-08-07

Nodulation formation efficiency D (NfeD) is a member of a class of membrane-anchored ClpP-class proteases. There is a second class of NfeD homologs that lack the ClpP domain. The genes of both NfeD classes usually are part of an operon that also contains a gene for a prokaryotic homolog of stomatin. (Stomatin is a major integral-membrane protein of mammalian erythrocytes.) Such NfeD/stomatin homolog gene pairs are present in more than 290 bacterial and archaeal genomes and their protein products may be part of the machinery used for quality control of membrane proteins. Herein, we report the structure of the isolated C-terminal domain of PH0471, a Pyrococcus horikoshii NfeD homolog, which lacks the ClpP domain. This C-terminal domain (termed NfeDC) contains a five-strand β-barrel, which is structurally very similar to the OB-fold (oligosaccharide/oligonucleotide–binding fold) domain. However, there is little sequence similarity between it and previously characterized OB-fold domains. The NfeDC domain lacks the conserved surface residues that are necessary for the binding of an OB-fold domain to DNA/RNA, an ion. Instead, its surface is composed of residues that are uniquely conserved in NfeD homologs and that form the structurally-conserved surface turns and β-bulges. There is also a conserved tryptophan present on the surface. We propose that, in general, NfeDC domains may interact with other spatially proximal membrane proteins and thereby regulate their activities.