Protein Science current issue

[ARTICLES] Crystallographic and biochemical studies revealing the structural basis for antizyme inhibitor function

Albeck, S., Dym, O., Unger, T., Snapir, Z., Bercovich, Z., Kahana, C. — 2008-04-24

Antizyme inhibitor (AzI) regulates cellular polyamine homeostasis by binding to the polyamine-induced protein, Antizyme (Az), with greater affinity than ornithine decarboxylase (ODC). AzI is highly homologous to ODC but is not enzymatically active. In order to understand these specific characteristics of AzI and its differences from ODC, we determined the 3D structure of mouse AzI to 2.05 Å resolution. Both AzI and ODC crystallize as a dimer. However, fewer interactions at the dimer interface, a smaller buried surface area, and lack of symmetry of the interactions between residues from the two monomers in the AzI structure suggest that this dimeric structure is nonphysiological. In addition, the absence of residues and interactions required for pyridoxal 5'-phosphate (PLP) binding suggests that AzI does not bind PLP. Biochemical studies confirmed the lack of PLP binding and revealed that AzI exists as a monomer in solution while ODC is dimeric. Our findings that AzI exists as a monomer and is unable to bind PLP provide two independent explanations for its lack of enzymatic activity and suggest the basis for its enhanced affinity toward Az.

[ARTICLES] N15 Cro and {lambda} Cro: Orthologous DNA-binding domains with completely different but equally effective homodimer interfaces

2008-04-24

Bacteriophage Cro proteins bind to target DNA as dimers but do not all dimerize with equal strength, and differ in fold in the region of the dimer interface. We report the structure of the Cro protein from Enterobacteria phage N15 at 1.05 Å resolution. The subunit fold contains five -helices and is closely similar to the structure of P22 Cro (1.3 Å backbone room mean square difference over 52 residues), but quite different from that of Cro, a structurally diverged member of this family with a mixed -helix/β-sheet fold. N15 Cro crystallizes as a biological dimer with an extensive interface (1303 Å² change in accessible surface area per dimer) and also dimerizes in solution with a K_d of 5.1 ± 1.5 µM. Its dimerization is much stronger than that of its structural homolog P22 Cro, which does not self-associate detectably in solution. Instead, the level of self-association and interfacial area for N15 Cro is similar to that of Cro, even though these two orthologs do not share the same fold and have dimer interfaces that are qualitatively different in structure. The common Cro ancestor is thought to be an all-helical monomer similar to P22 Cro. We propose that two Cro descendants independently developed stronger dimerization by entirely different mechanisms.

[ARTICLES] Structure and ion channel activity of the human respiratory syncytial virus (hRSV) small hydrophobic protein transmembrane domain

Gan, S. W., Ng, L., Lin, X., Gong, X., Torres, J. — 2008-04-24

The small hydrophobic (SH) protein from the human respiratory syncytial virus (hRSV) is a glycoprotein of ~64 amino acids with one putative -helical transmembrane domain. Although SH protein is important for viral infectivity, its exact role during viral infection is not clear. Herein, we have studied the secondary structure, orientation, and oligomerization of the transmembrane domain of SH (SH-TM) in the presence of lipid bilayers. Only one oligomer, a pentamer, was observed in PFO-PAGE. Using polarized attenuated total reflection-Fourier transform infrared (PATR-FTIR) spectroscopy, we show that the SH-TM is -helical. The rotational orientation of SH-TM was determined by site-specific infrared dichroism (SSID) at two consecutive isotopically labeled residues. This orientation is consistent with that of an evolutionary conserved pentameric model obtained from a global search protocol using 13 homologous sequences of RSV. Conductance studies of SH-TM indicate ion channel activity, which is cation selective, and inactive below the predicted pK_a of histidine. Thus, our results provide experimental evidence that the transmembrane domain of SH protein forms pentameric -helical bundles that form cation-selective ion channels in planar lipid bilayers. We provide a model for this pore, which should be useful in mutagenesis studies to elucidate its role during the virus cycle.

[ARTICLES] Structure and dynamics of de novo proteins from a designed superfamily of 4-helix bundles

Go, A., Kim, S., Baum, J., Hecht, M. H. — 2008-04-24

Libraries of de novo proteins provide an opportunity to explore the structural and functional potential of biological molecules that have not been biased by billions of years of evolutionary selection. Given the enormity of sequence space, a rational approach to library design is likely to yield a higher fraction of folded and functional proteins than a stochastic sampling of random sequences. We previously investigated the potential of library design by binary patterning of hydrophobic and hydrophilic amino acids. The structure of the most stable protein from a binary patterned library of de novo 4-helix bundles was solved previously and shown to be consistent with the design. One structure, however, cannot fully assess the potential of the design strategy, nor can it account for differences in the stabilities of individual proteins. To more fully probe the quality of the library, we now report the NMR structure of a second protein, S-836. Protein S-836 proved to be a 4-helix bundle, consistent with design. The similarity between the two solved structures reinforces previous evidence that binary patterning can encode stable, 4-helix bundles. Despite their global similarities, the two proteins have cores that are packed at different degrees of tightness. The relationship between packing and dynamics was probed using the Modelfree approach, which showed that regions containing a high frequency of chemical exchange coincide with less well-packed side chains. These studies show (1) that binary patterning can drive folding into a particular topology without the explicit design of residue-by-residue packing, and (2) that within a superfamily of binary patterned proteins, the structures and dynamics of individual proteins are modulated by the identity and packing of residues in the hydrophobic core.

[ARTICLES] A buried lysine that titrates with a normal pKa : Role of conformational flexibility at the protein-water interface as a determinant of pKa values

2008-04-24

Previously we reported that Lys, Asp, and Glu residues at positions 66 and 92 in staphylococcal nuclease (SNase) titrate with pK_a values shifted by up to 5 pK_a units in the direction that promotes the neutral state. In contrast, the internal Lys-38 in SNase titrates with a normal pK_a . The crystal structure of the L38K variant shows that the side chain of Lys-38 is buried. The ionizable moiety is ~7 Å from solvent and ion paired with Glu-122. This suggests that the pK_a value of Lys-38 is normal because the energetic penalty for dehydration is offset by a favorable Coulomb interaction. However, the pK_a of Lys-38 was also normal when Glu-122 was replaced with Gln or with Ala. Continuum electrostatics calculations were unable to reproduce the pK_a of Lys-38 unless the protein was treated with an artificially high dielectric constant, consistent with structural reorganization being responsible for the normal pK_a value of Lys-38. This reorganization must be local because circular dichroism and NMR spectroscopy indicate that the L38K protein is native-like under all conditions studied. In molecular dynamics simulations, the ion pair between Lys-38 and Glu-122 is unstable. The simulations show that a minor rearrangement of a loop is sufficient to allow penetration of water to the amino moiety of Lys-38. This illustrates both the important roles of local flexibility and water penetration as determinants of pK_a values of ionizable groups buried near the protein–water interface, and the challenges faced by structure-based pK_a calculations in reproducing these effects.

[ARTICLES] Cysteine 155 plays an important role in the assembly of Mycobacterium tuberculosis FtsZ

Jaiswal, R., Panda, D. — 2008-04-24

The assembly of FtsZ plays an important role in bacterial cell division. Mycobacterium tuberculosis FtsZ (MtbFtsZ) has a single cysteine residue at position 155. We have investigated the role of the lone cysteine residue in the assembly of MtbFtsZ using different complimentary approaches, namely chemical modification by a thiol-specific reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) or a cysteine-chelating agent HgCl₂, and site-directed mutagenesis of the cysteine residue. HgCl₂ strongly reduced the polymerized mass of MtbFtsZ while it had no detectable effect on the polymerization of Escherichia coli FtsZ, which lacks a cysteine residue. HgCl₂ inhibited the protofilamentous assembly of MtbFtsZ and induced the aggregation of the protein. Further, HgCl₂ perturbed the secondary structure of MtbFtsZ and increased the binding of a hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) with MtbFtsZ, indicating that the binding of HgCl₂ altered the conformation of MtbFtsZ. Chemical modification of MtbFtsZ by DTNB also decreased the polymerized mass of MtbFtsZ. Further, the mutagenesis of Cys-155 to alanine caused a strong reduction in the assembly of MtbFtsZ. Under assembly conditions, the mutated protein formed aggregates instead of protofilaments. Far-UV CD spectroscopy and ANS binding suggested that the mutated MtbFtsZ has different conformation than that of the native MtbFtsZ. The effect of the mutation or chemical modification of Cys-155 on the MtbFtsZ assembly has been explained considering its location in the MtbFtsZ crystal structure. The results together suggest that the cysteine residue (Cys-155) of MtbFtsZ plays an important role in the assembly of MtbFtsZ into protofilaments.

[ARTICLES] Structural effects of Parkinson's disease linked DJ-1 mutations

Malgieri, G., Eliezer, D. — 2008-04-24

Mutations in the protein DJ-1 are associated with familial forms of Parkinson's disease, indicating that DJ-1 may be involved in pathways related to the etiology of this disorder. Here we have used solution state NMR and circular dichroism spectroscopies to evaluate the extent of structural perturbations associated with five different Parkinson's disease linked DJ-1mutations: L166P, E64D, M26I, A104T, and D149A. Comparison of the data with those obtained for the wild-type protein shows that the L166P mutation leads to severe and global destabilization and unfolding of the protein structure, while the structure of the E64D mutation, as expected, is nearly unperturbed. Interestingly, the remaining three mutants all show different degrees of structural perturbation, which are accompanied by a reduction in the thermodynamic stability of the protein. The observed structural and thermodynamic differences are likely to underlie any functional variations between these mutants and the wild type, which in turn are likely responsible for the pathogenicity of these mutations.

[ARTICLES] Structure and ligand binding of the soluble domain of a Thermotoga maritima membrane protein of unknown function TM1634

McCleverty, C. J., Columbus, L., Kreusch, A., Lesley, S. A. — 2008-04-24

As a part of the Joint Center for Structural Genomics (JCSG) biological targets, the structures of soluble domains of membrane proteins from Thermotoga maritima were pursued. Here, we report the crystal structure of the soluble domain of TM1634, a putative membrane protein of 128 residues (15.1 kDa) and unknown function. The soluble domain of TM1634 is an -helical dimer that contains a single tetratrico peptide repeat (TPR) motif in each monomer where each motif is similar to that found in Tom20. The overall fold, however, is unique and a DALI search does not identify similar folds beyond the 38-residue TPR motif. Two different putative ligand binding sites, in which PEG200 and Co²⁺ were located, were identified using crystallography and NMR, respectively.

[ARTICLES] Structural basis for the NAD-hydrolysis mechanism and the ARTT-loop plasticity of C3 exoenzymes

Menetrey, J., Flatau, G., Boquet, P., Menez, A., Stura, E. A. — 2008-04-24

C3-like exoenzymes are ADP-ribosyltransferases that specifically modify some Rho GTPase proteins, leading to their sequestration in the cytoplasm, and thus inhibiting their regulatory activity on the actin cytoskeleton. This modification process goes through three sequential steps involving NAD-hydrolysis, Rho recognition, and binding, leading to Rho ADP-ribosylation. Independently, three distinct residues within the ARTT loop of the C3 exoenzymes are critical for each of these steps. Supporting the critical role of the ARTT loop, we have shown previously that it adopts a distinct conformation upon NAD binding. Here, we present seven wild-type and ARTT loop-mutant structures of C3 exoenzyme of Clostridium botulinum free and bound to its true substrate, NAD, and to its NAD-hydrolysis product, nicotinamide. Altogether, these structures expand our understanding of the conformational diversity of the C3 exoenzyme, mainly within the ARTT loop.

[ARTICLES] Molecular determinants of the aggregation behavior of {alpha}- and {beta}-synuclein

2008-04-24

- and β-synuclein are closely related proteins, the first of which is associated with deposits formed in neurodegenerative conditions such as Parkinson's disease while the second appears to have no relationship to any such disorders. The aggregation behavior of - and β-synuclein as well as a series of chimeric variants were compared by exploring the structural transitions that occur in the presence of a widely used lipid mimetic, sodium dodecyl sulfate (SDS). We found that the aggregation rates of all these protein variants are significantly enhanced by low concentrations of SDS. In particular, we inserted the 11-residue sequence of mainly hydrophobic residues from the non-amyloid-β-component (NAC) region of -synuclein into β-synuclein and show that the fibril formation rate of this chimeric protein is only weakly altered from that of β-synuclein. These intrinsic propensities to aggregate are rationalized to a very high degree of accuracy by analysis of the sequences in terms of their associated physicochemical properties. The results begin to reveal that the differences in behavior are primarily associated with a delicate balance between the positions of a range of charged and hydrophobic residues rather than the commonly assumed presence or absence of the highly aggregation-prone region of the NAC region of -synuclein. This conclusion provides new insights into the role of -synuclein in disease and into the factors that regulate the balance between solubility and aggregation of a natively unfolded protein.

[ARTICLES] RDC-assisted modeling of symmetric protein homo-oligomers

Wang, X., Bansal, S., Jiang, M., Prestegard, J. H. — 2008-04-24

Protein oligomerization serves an important function in biological processes, yet solving structures of protein oligomers has always been a challenge. For solution NMR, the challenge arises both from the increased size of these systems and, in the case of homo-oligomers, from ambiguities in assignment of intra- as opposed to intersubunit NOEs. In this study, we present a residual dipolar coupling (RDC)-assisted method for constructing models of homo-oligomers with purely rotational symmetry. Utilizing the fact that one of the principal axes of the tensor describing the alignment needed for RDC measurement is always parallel to the oligomer symmetry axis, it is possible to greatly restrict possible models for the oligomer. Here, it is shown that, if the monomer structure is known, all allowed dimer models can be constructed using a grid search algorithm and evaluated based on RDC simulations and the quality of the interface between the subunits. Using the Bacillus subtilis protein YkuJ as an example, it is shown that the evaluation criteria based on just two sets of NH RDCs are very selective and can unambiguously produce a model in good agreement with an existing X-ray structure of YkuJ.

[ARTICLES] Miranda cargo-binding domain forms an elongated coiled-coil homodimer in solution: Implications for asymmetric cell division in Drosophila

Yousef, M. S., Kamikubo, H., Kataoka, M., Kato, R., Wakatsuki, S. — 2008-04-24

Miranda is a multidomain adaptor protein involved in neuroblast asymmetric division in Drosophila melanogaster. The central domain of Miranda is necessary for cargo binding of the neural transcription factor Prospero, the Prospero-mRNA carrier Staufen, and the tumor suppressor Brat. Here, we report the first solution structure of Miranda central "cargo-binding" domain (residues 460–660) using small-angle X-ray scattering. Ab initio modeling of the scattering data yields an elongated "rod-like" molecule with a maximum linear dimension (D_max) of ~22 nm. Moreover, circular dichroism and cross-linking experiments indicate that the cargo-binding domain is predominantly helical and forms a parallel coiled-coil homodimer in solution. Based on the results, we modeled the full-length Miranda protein as a double-headed, double-tailed homodimer with a long central coiled-coil region. We discuss the cargo-binding capacity of the central domain and propose a structure-based mechanism for cargo release and timely degradation of Miranda in developing neuroblasts.

[ARTICLES] Correspondences between low-energy modes in enzymes: Dynamics-based alignment of enzymatic functional families

Zen, A., Carnevale, V., Lesk, A. M., Micheletti, C. — 2008-04-24

Proteins that show similarity in their equilibrium dynamics can be aligned by identifying regions that undergo similar concerted movements. These movements are computed from protein native structures using coarse-grained elastic network models. We show the existence of common large-scale movements in enzymes selected from the main functional and structural classes. Alignment via dynamics does not require prior detection of sequence or structural correspondence. Indeed, a third of the statistically significant dynamics-based alignments involve enzymes that lack substantial global or local structural similarities. The analysis of specific residue–residue correspondences of these structurally dissimilar enzymes in some cases suggests a functional relationship of the detected common dynamic features. Including dynamics-based criteria in protein alignment thus provides a promising avenue for relating and grouping enzymes in terms of dynamic aspects that often, though not always, assist or accompany biological function.

[ARTICLES] Transmembrane helices that form two opposite homodimeric interactions: An asparagine scan study of {alpha}M and {beta}2 integrins

Parthasarathy, K., Lin, X., Tan, S. M., Law, S.K. A., Torres, J. — 2008-04-24

Integrins are /β heterodimers, but recent in vitro and in vivo experiments also suggest an ability to associate through their transmembrane domains to form homomeric interactions. While the results of some in vitro experiments are consistent with an interaction mediated by a GxxxG-like motif, homo-oligomers observed after in vivo cross-linking are consistent with an almost opposite helix–helix interface. We have shown recently that both models of interaction are compatible with evolutionary conservation data, and we predicted that the -helices in both models would have a similar rotational orientation. Herein, we have tested our prediction using in vitro asparagine scan of five consecutive residues along the GxxxG-like motif of the transmembrane domain of and β integrins, M and β2. We show that Asn-mediated dimerization occurs twice for every turn of the helix, consistent with two almost opposite forms of interaction as suggested previously for IIb and β3 transmembrane domains. The orientational parameters helix tilt and rotational orientation of each of these two Asn-stabilized dimers were measured by site-specific infrared dichroism (SSID) in model lipid bilayers and were found to be consistent with our predicted computational models. Our results highlight an intrinsic tendency for integrin transmembrane -helices to form two opposite types of homomeric interaction in addition to their heteromeric interactions and suggest that integrins may form complex and specific networks at the transmembrane domain during function.

[PROTEIN STRUCTURE REPORTS] Conformational gating of dimannose binding to the antiviral protein cyanovirin revealed from the crystal structure at 1.35 A resolution

Fromme, R., Katiliene, Z., Fromme, P., Ghirlanda, G. — 2008-04-24

Cyanovirin (CV-N) is a small lectin with potent HIV neutralization activity, which could be exploited for a mucosal defense against HIV infection. The wild-type (wt) protein binds with high affinity to mannose-rich oligosaccharides on the surface of gp120 through two quasi-symmetric sites, located in domains A and B. We recently reported on a mutant of CV-N that contained a single functional mannose-binding site, domain B, showing that multivalent binding to oligomannosides is necessary for antiviral activity. The structure of the complex with dimannose determined at 1.8 Å resolution revealed a different conformation of the binding site than previously observed in the NMR structure of wt CV-N. Here, we present the 1.35 Å resolution structure of the complex, which traps three different binding conformations of the site and provides experimental support for a locking and gating mechanism in the nanoscale time regime observed by molecular dynamics simulations.

[FOR THE RECORD] The C3 domain of Pasteurella multocida toxin is the minimal domain responsible for activation of Gq-dependent calcium and mitogenic signaling

Aminova, L. R., Luo, S., Bannai, Y., Ho, M., Wilson, B. A. — 2008-04-24

The large 1285-amino-acid protein toxin from Pasteurella multocida (PMT) is a multifunctional single-chain polypeptide that binds to and enters eukaryotic cells and acts intracellularly to promote G_q and G_12/13 protein-dependent calcium and mitogenic signal transduction. Previous studies indicated that the intracellular activity domain responsible for PMT action was located within the C-terminal 600–700 amino acids. In this study, we have exogenously expressed a series of N- and C-terminal PMT fragments directly in mammalian cells and have used the dual luciferase reporter system to assay for toxin-mediated activation of calcium-calcineurin-NFAT signaling (NFAT-luciferase) and mitogenic serum response signaling (SRE-luciferase). Using this approach, we have defined the last 180 amino acids, which encompass the C3 domain in the crystal structure, as the minimum domain sufficient to activate both NFAT and SRE signaling pathways.

[FOR THE RECORD] Correlation between 13C {alpha} chemical shifts and helix content of peptide ensembles

Weinstock, D. S., Narayanan, C., Baum, J., Levy, R. M. — 2008-04-24

Replica exchange molecular dynamics simulations are used to generate three ensembles of an S-peptide analog (AETAAAKFLREHMDS). Percent helicity of the peptide ensembles calculated using STRIDE is compared to percent helicity calculated from ¹³C chemical shift deviations (CSD) from random coil in order to test the assumption that CSD can be correlated to percent helicity. The two estimates of helicity, one based on structure and the other on CSD, are in close to quantitative agreement, except at the edges of helical stretches where disagreements of as much as 50% can be found. These disagreements can occur by CSDs both as an under- and an overestimate of peptide helicity. We show that underestimation arises due to ensemble averaging of positive CSDs from conformers with torsion angles in the helical region of Ramachandran space with negative CSDs corresponding to conformers of the peptide in the extended region. In contrast, overestimation comes about due to the fact that a large number of conformations with torsion angles in the helical region are not counted as helical by STRIDE due to a lack of correlated helical torsion angles in neighboring residues.

[FOR THE RECORD] Contribution of dipole-dipole interactions to the stability of the collagen triple helix

Improta, R., Berisio, R., Vitagliano, L. — 2008-04-24

Unveiling sequence–stability and structure–stability relationships is a major goal of protein chemistry and structural biology. Despite the enormous efforts devoted, answers to these issues remain elusive. In principle, collagen represents an ideal system for such investigations due to its simplified sequence and regular structure. However, the definition of the molecular basis of collagen triple helix stability has hitherto proved to be a difficult task. Particularly puzzling is the decoding of the mechanism of triple helix stabilization/destabilization induced by imino acids. Although the propensity-based model, which correlates the propensities of the individual imino acids with the structural requirements of the triple helix, is able to explicate most of the experimental data, it is unable to predict the rather high stability of peptides embedding Gly–Hyp–Hyp triplets. Starting from the available X-ray structures of this polypeptide, we carried out an extensive quantum chemistry analysis of the mutual interactions established by hydroxyproline residues located at the X and Y positions of the Gly–X–Y motif. Our data clearly indicate that the opposing rings of these residues establish significant van der Waals and dipole–dipole interactions that play an important role in triple helix stabilization. These findings suggest that triple helix stabilization can be achieved by distinct structural mechanisms. The interplay of these subtle but recurrent effects dictates the overall stability of this widespread structural motif.

[BOOK REVIEW] Cell-free protein synthesis: Methods and protocols, edited by Alexander S. Spirin and James R. Swartz.

Savage, D. F. — 2008-04-24