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1.  D-polyglutamine amyloid recruits L-polyglutamine monomers and kills cells 
Journal of molecular biology  2013;426(4):816-829.
Polyglutamine (polyQ) amyloid fibrils are observed in disease tissue and have been implicated as toxic agents responsible for neurodegeneration in expanded CAG repeat diseases like Huntington’s disease (HD). Despite intensive efforts, the mechanism of amyloid toxicity remains unknown. As a novel approach to probing polyQ toxicity, we investigate here how some cellular and physical properties of polyQ amyloid vary with the chirality of the glutamine residues in the polyQ. We challenged PC12 cells with small amyloid fibrils composed of either L- or D-polyQ peptides and found that D-fibrils are as cytotoxic as L-fibrils. We also found using fluorescence microscopy that both aggregates effectively seed the aggregation of cell-produced L-polyQ proteins, suggesting a surprising lack of stereochemical restriction in seeded elongation of polyQ amyloid. To investigate this effect further, we studied chemically synthesized D- and L-polyQ in vitro. We found that, as expected, D-polyQ monomers are not recognized by proteins that recognize L-polyQ monomers. However, amyloid fibrils prepared from D-polyQ peptides can efficiently seed the aggregation of L-polyQ monomers in vitro, and vice versa. This result is consistent with our cell results on polyQ recruitment, but is inconsistent with previous literature reports on the chiral specificity of amyloid seeding. This chiral cross-seeding can be rationalized by a model for seeded elongation featuring a “rippled β-sheet” interface between seed fibril and docked monomers of opposite chirality. The lack of chiral discrimination in polyQ amyloid cytotoxicity is consistent with several toxicity mechanisms, including recruitment of cellular polyQ proteins.
PMCID: PMC3910295  PMID: 24291210
chiral cross-seeding; rippled β-sheet; toxicity; recruitment; Huntington’s disease
2.  PFKFB3 regulates oxidative stress homeostasis via its S-glutathionylation in cancer 
Journal of molecular biology  2013;426(4):830-842.
Whereas moderately increased cellular oxidative stress is supportive for cancerous growth of cells, excessive levels of reactive oxygen species (ROS) is detrimental to their growth and survival. We demonstrated that high ROS levels, via increased oxidized glutathione (GSSG), induce isoform-specific S-glutathionylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) at the residue Cys206, which is located near the entrance to the 6-phosphofructo-2-kinase catalytic pocket. Upon this ROS-dependent, reversible, covalent modification, a marked decrease in its catalytic ability to synthesize the Fructose-2,6-bisphosphate (Fru-2,6-P2), the key glycolysis allosteric activator, was observed. This event was coupled to a decrease in glycolytic flux and an increase in glucose metabolic flux into the pentose phosphate pathway (PPP). This shift, in turn, caused an increase in reduced glutathione (GSH) and, ultimately, resulted in ROS detoxification inside HeLa cells. The ability of PFKFB3 to control the Fru-2,6-P2 levels in an ROS-dependent manner allows the PFKFB3-expressing cancer cells to continue energy metabolism with a reduced risk of excessive oxidative stress and, thereby, to support their cell survival and proliferation. This study provides a new insight into the roles of PFKFB3 as switch that senses and controls redox homeostasis in cancer in addition to its role in cancer glycolysis.
PMCID: PMC3919529  PMID: 24295899
reactive oxygen species; Warburg effect; TCA cycle; glutathione; pentose phosphate pathway
3.  Functional clustering of immunoglobulin superfamily proteins with protein-protein interaction information calibrated Hidden Markov model sequence-profiles 
Journal of molecular biology  2013;426(4):945-961.
Secreted and cell surface-localized members of the immunoglobulin superfamily (IgSF) play central roles in regulating adaptive and innate immune responses, and are prime targets for the development of protein-based therapeutics. An essential activity of the ectodomains of these proteins is the specific recognition of cognate ligands, which are often other members of the IgSF. In this work we provide functional insight for this important class of proteins through the development of a clustering algorithm that groups together extracellular domains of the IgSF with similar binding preferences. Information from hidden Markov model-based sequence profiles and domain structure is calibrated against manually curated protein interaction data to define functional families of IgSF proteins. The method is able to assign 82% of the 477 extracellular IgSF protein to a functional family, while the rest are either single proteins with unique function or proteins that could not be assigned with the current technology. The functional clustering of IgSF proteins generates hypotheses regarding the identification of new cognate receptor:ligand pairs and reduces the pool of possible interacting partners to a manageable level for experimental validation.
PMCID: PMC3946809  PMID: 24246499
Immunoglobulin superfamily; protein-protein interaction; functional prediction
4.  The glaucoma-associated olfactomedin domain of myocilin forms polymorphic fibrils that are constrained by partial unfolding and peptide sequence 
Journal of molecular biology  2013;426(4):921-935.
The glaucoma-associated olfactomedin domain of myocilin (myoc-OLF) is a recent addition to the growing list of disease-associated amyloidogenic proteins. Inherited, diseasecausing myocilin variants aggregate intracellularly instead of being secreted to the trabecular meshwork (TM), which is a scenario toxic to TM cells and leads to early onset of ocular hypertension, the major risk factor for glaucoma. Here we systematically structurally and biophysically dissected myoc-OLF to better understand its amyloidogenesis. Under mildly destabilizing conditions, wild-type myoc-OLF adopts non-native structures that readily fibrillize when incubated at a temperature just below the transition for tertiary unfolding. With buffers at physiological pH, two main end-point fibril morphologies are observed: (a) straight fibrils common to many amyloids and (b) unique micron-length, ~300 nm or larger diameter species that lasso oligomers, which also exhibit classical spectroscopic amyloid signatures. Three disease-causing variants investigated herein exhibit non-native tertiary structures under physiological conditions, leading to accelerated growth rates and a variety of fibril morphologies. In particular, the well-documented D380A variant, which lacks calcium, forms large circular fibrils. Two amyloid forming peptide stretches have been identified, one for each of the main fibril morphologies observed. Our study places myoc-OLF within the larger landscape of the amylome and provides insight into the diversity of myoc-OLF aggregation that plays a role in glaucoma pathogenesis.
PMCID: PMC3946817  PMID: 24333014
Amyloid; protein misfolding; protein structure; circular dichroism; atomic force microscopy
5.  The Crystal Structure of the Hinge Domain of the Escherichia coli Structural Maintenance of Chromosomes Protein MukB 
Journal of molecular biology  2009;395(1):11-19.
MukB, a divergent structural maintenance of chromosomes (SMC) protein, is important for chromosomal segregation and condensation in γ-proteobacteria. MukB and canonical SMC proteins share a characteristic five-domain structure. Globular N- and C-terminal domains interact to form an ABC-like ATPase or “head” domain, which is connected to a smaller dimerization or “hinge” domain by a long, antiparallel coiled coil. In addition to mediating dimerization, this hinge region has been implicated in both conformational flexibility and dynamic protein-DNA interactions. We report here the first crystallographic model of the MukB hinge domain. This model also contains approximately 20% of the coiled coil domain, including an unusual coiled coil deviation. These results will facilitate studies to clarify the roles of both the hinge and coiled coil domains in MukB function.
PMCID: PMC4335309  PMID: 19853611
MukB; Structural Maintenance of Chromosomes (SMC); hinge; coiled coil; condensin
6.  Efficient Recovery of High-Affinity Antibodies from a Single-Chain Fab Yeast Display Library 
Journal of molecular biology  2009;389(2):365-375.
Yeast display is a powerful technology for the isolation of monoclonal antibodies (mAbs) against a target antigen. Antibody libraries have been displayed on the surface of yeast as both single-chain variable fragment (scFv) and antigen binding fragment (Fab). Here, we combine these two formats to display well-characterized mAbs as single-chain Fabs (scFabs) on the surface of yeast and construct the first scFab yeast display antibody library. When expressed on the surface of yeast, two out of three anti-human immunodeficiency virus (HIV)-1 mAbs bound with higher affinity as scFabs than scFvs. Also, the soluble scFab preparations exhibited binding and neutralization profiles comparable to that of the corresponding Fab fragments. Display of an immune HIV-1 scFab library on the surface of yeast, followed by rounds of sorting against HIV-1 gp120, allowed for the selection of 13 antigen-specific clones. When the same cDNA was used to construct the library in an scFv format, a similar number but a lower affinity set of clones were selected. Based on these results, yeast-displayed scFab libraries can be constructed and selected with high efficiency, characterized without the need for a reformatting step, and used to isolate higher-affinity antibodies than scFv libraries.
PMCID: PMC4332843  PMID: 19376130
immune repertoire; antibody display format; HIV-1; flow cytometry; antibody library
7.  Intestinal MUC2 mucin supramolecular topology by packing and release resting on D3 domain assembly 
Journal of molecular biology  2014;426(14):2567-2579.
MUC2 is the major gel-forming mucin of the colon forming a protective gel barrier organized into an inner stratified and an outer loose layer. The MUC2 N-terminus (D1-D2-D'D3 domains) has a dual function in building a net-like structure by disulfide-bonded trimerization and packing the MUC2 polymer into an N-terminal concatenated polygonal platform with the C-termini extending perpendicularly by pH- and calcium-dependent interactions. We studied the N-terminal D'D3 domain by producing three recombinant variants, with or without Myctag and GFP, and analyzed these by gel filtration, electron microscopy and single particle image processing. The three variants were all trimers when analyzed upon denaturing conditions, but eluted as hexamers upon gel filtration under native conditions. Studies by electron microscopy and 3D maps revealed cage-like structures with two- and three-fold symmetries. The structure of the MUC2 D3 domain confirms that the MUC2 mucin forms branched net-like structures. This suggests that the MUC2 mucin is stored with two N-terminal concatenated ring platforms turned by 180° against each other implicating that every second unfolded MUC2 net in mature mucus is turned upside-down.
PMCID: PMC4293034  PMID: 24816392
mucin; structure; electron microscopy; cystic fibrosis; intestine
8.  Structural Studies of Streptococcus pyogenes Streptolysin O Provide Insights into the Early Steps of Membrane Penetration 
Journal of molecular biology  2013;426(4):785-792.
Cholesterol-dependent cytolysins (CDCs) are a large family of bacterial toxins that exhibit a dependence on the presence of membrane cholesterol in forming large pores in cell membranes. Significant changes in the three-dimensional structure of these toxins are necessary to convert the soluble monomeric protein into a membrane pore. We have determined the crystal structure of the archetypical member of the CDC family, streptolysin O (SLO), a virulence factor from Streptococcus pyogenes. The overall fold is similar to previously reported CDC structures, although the C-terminal domain is in a different orientation with respect to the rest of the molecule. Surprisingly, a signature stretch of CDC sequence called the undecapeptide motif, a key region involved in membrane recognition, adopts a very different structure in SLO to that of the well-characterized CDC perfringolysin O (PFO), although the sequences in this region are identical. An analysis reveals that, in PFO, there are complementary interactions between the motif and the rest of domain 4 that are lost in SLO. Molecular dynamics simulations suggest that the loss of a salt bridge in SLO and a cation–pi interaction are determining factors in the extended conformation of the motif, which in turn appears to result in a greater flexibility of the neighboring L1 loop that houses a cholesterol-sensing motif. These differences may explain the differing abilities of SLO and PFO to efficiently penetrate target cell membranes in the first step of toxin insertion into the membrane.
PMCID: PMC4323271  PMID: 24316049
conformational change; membrane insertion; molecular dynamics simulations; pore-forming toxins; X-ray crystallography
9.  A LIM-9 (FHL) / SCPL-1 (SCP) Complex Interacts with the C-terminal Protein Kinase Regions of UNC-89 (Obscurin) in C. elegans Muscle 
Journal of molecular biology  2009;386(4):976-988.
The C. elegans gene unc-89 encodes a set of mostly giant polypeptides (up to 900 kDa) that contain multiple Ig and Fn3, a triplet of SH3-DH-PH, and two protein kinase domains. The loss of function mutant phenotype and localization of antibodies to UNC-89 proteins, indicate that the function of UNC-89 is to help organize sarcomeric A-bands, especially M-lines. Recently, we reported that each of the protein kinase domains interact with SCPL-1, which contains a CTD type protein phosphatase domain. Here, we report that SCPL-1 interacts with LIM-9 (FHL), a protein that we first discovered as an interactor of UNC-97 (PINCH) and UNC-96, components of an M-line costamere in nematode muscle. We also show that LIM-9 can interact with UNC-89 through its first kinase domain and a portion of unique sequence lying between the two kinase domains. All the interactions were confirmed by biochemical methods. A yeast three-hybrid assay demonstrates a ternary complex between the two protein kinase regions and SCPL-1. Evidence that the UNC-89/SCPL-1 interaction occurs in vivo was provided by showing that overexpression of SCPL-1 results in disorganization of UNC-89 at M-lines. We suggest two structural models for the interactions of SCPL-1 and LIM-9 with UNC-89 at the M-line.
PMCID: PMC4321865  PMID: 19244614
C. elegans; obscurin; FHL; CTD phosphatase; muscle
10.  Protein-mediated chromosome pairing of repetitive arrays 
Journal of molecular biology  2013;426(3):550-557.
Chromosomally-integrated arrays of lacO and tetO operator sites visualized by LacI and TetR repressor proteins fused with GFP (or other fluorescent proteins) are widely used to monitor the behavior of chromosomal loci in various systems. Yet, insertion of such arrays and expression of the corresponding proteins is known to perturb genomic architecture. In several cases, juxtaposition of such arrays located on different chromosomes has been inferred to reflect pairing of the corresponding loci. Here, we report that a version of TetR-GFP mutated to disrupt GFP dimerization (TetR-A206KGFP or “TetR-kGFP”), abolishes pairing of tetO-arrays in vivo and brings spatial proximity of chromosomal loci marked with those arrays back to the wild type level. These data argue that pairing of arrays is caused by GFP dimerization and thus presents an example of protein-assisted interaction in chromosomes. Arrays marked with another protein, TetR-tdTomato, which has a propensity to form intra-molecular dimers instead of inter-molecular dimers, also display reduced level of pairing, supporting this idea. TetR-kGFP provides an improved system for studying chromosomal loci with a low pairing background.
PMCID: PMC3898463  PMID: 24211468
11.  The oligomeric state of the active Vps4 AAA ATPase 
Journal of molecular biology  2013;426(3):510-525.
The cellular ESCRT pathway drives membrane constriction toward the cytosol and effects membrane fission during cytokinesis, endosomal sorting, and the release of many enveloped viruses, including HIV. A component of this pathway, the AAA ATPase Vps4, provides energy for pathway progression. Although it is established that Vps4 functions as an oligomer, subunit stoichiometry and other fundamental features of the functional enzyme are unclear. Higher-order oligomers have thus far only been characterized for a Walker B mutant of Vps4 in the presence of ATP. Here, we report that although some mutant Vps4 proteins form dodecameric assemblies, active wild-type S. cerevisiae and S. solfataricus Vps4 enzymes can form hexamers in the presence of ATP and ADP, as assayed by size exclusion chromatography and equilibrium analytical ultracentifugation. The Vta1p activator binds hexameric yeast Vps4p without changing the oligomeric state of Vps4p, implying that the active Vta1p:Vps4p complex also contains a single hexameric ring. Additionally, we report crystal structures of two different archaeal Vps4 homologs, whose structures and lattice interactions suggest a conserved mode of oligomerization. Disruption of the proposed hexamerization interface by mutagenesis abolished the ATPase activity of archaeal Vps4 proteins and blocked Vps4p function in S. cerevisiae. These data challenge the prevailing model that active Vps4 is a double ring dodecamer, and argue that, like other type I AAA ATPases, Vps4 functions as a single ring with six subunits.
PMCID: PMC3919030  PMID: 24161953
multivesicular body; HIV budding; ESCRT; AAA ATPase; protein oligomerization; X-ray crystallography
12.  Interaction of HoloCcmE with CcmF in Heme Trafficking and Cytochrome c Biosynthesis 
Journal of molecular biology  2014;426(3):570-585.
The periplasmic heme chaperone holoCcmE is essential for heme trafficking in the cytochrome c biosynthetic pathway in many bacteria, archaea, and plant mitochondria. This pathway, called system I, involves two steps: i) formation and release of holoCcmE (by the ABC-transporter complex CcmABCD), and ii) delivery of the heme in holoCcmE to the putative cytochrome c heme lyase complex, CcmFH. CcmFH is believed to facilitate the final covalent attachment of heme (from holoCcmE) to the apocytochrome c. Although most models for system I propose that holoCcmE delivers heme directly to CcmF, no interaction between holoCcmE and CcmF has been demonstrated. Here, a complex between holoCcmE and CcmF is “trapped”, purified, and characterized. HoloCcmE must be released from the ABC-transporter complex CcmABCD to interact with CcmF, and the holo-form of CcmE interacts with CcmF at levels at least 20-fold higher than apoCcmE. Two conserved histidines (here termed P-His1 and P-His2) in separate periplasmic loops in CcmF are required for interaction with holoCcmE, and evidence is presented that P-His1 and P-His2 function as heme-binding ligands. These results show that heme in holoCcmE is essential for complex formation with CcmF, and that the heme of holoCcmE is coordinated by P-His1 and P-His2 within the WWD domain of CcmF. These features are strikingly similar to formation of the CcmC:heme:CcmE ternary complex (Richard-Fogal and Kranz, JMB 2010), and suggest common mechanistic and structural aspects.
PMCID: PMC3943652  PMID: 24513106
Pathway; Biogenesis; Oxidation-Reduction; Heme trafficking; Cytochrome c Maturation
13.  Insights into eukaryotic primer synthesis from structures of the p48 subunit of human DNA primase 
Journal of molecular biology  2013;426(3):558-569.
DNA replication in all organisms requires polymerases to synthesize copies of the genome. DNA polymerases are unable to function on a bare template and require a primer. Primases are crucial RNA polymerases that perform the initial de novo synthesis, generating the first 8–10 nucleotides of the primer. Although structures of archaeal and bacterial primases have provided insights into general priming mechanisms, these proteins are not well conserved with heterodimeric (p48/p58) primases in eukaryotes. Here, we present X-ray crystal structures of the catalytic engine of a eukaryotic primase, which is contained in the p48 subunit. The structures of p48 reveal eukaryotic primases maintain the conserved catalytic prim fold domain, but with a unique sub-domain not found in the archaeal and bacterial primases. Calorimetry experiments reveal Mn2+ but not Mg2+ significantly enhances the binding of nucleotide to primase, which correlates with in vitro higher catalytic efficiency. The structure of p48 with bound UTP and Mn2+ provides insights into the mechanism of nucleotide synthesis by primase. Substitution of conserved residues involved in either metal or nucleotide binding altered nucleotide binding affinities, and yeast strains containing the corresponding Pri1p substitutions were not viable. Our results revealed two residues (S160 and H166) in direct contact with the nucleotide that were previously unrecognized as critical to the human primase active site. Comparing p48 structures to those of similar polymerases in different states of action suggests changes that would be required to attain a catalytically competent conformation capable of initiating dinucleotide synthesis.
PMCID: PMC3946992  PMID: 24239947
DNA replication; DNA primase; manganese; X-family DNA polymerase
14.  Structural complementation of the catalytic domain of Pseudomonas exotoxin A 
Journal of molecular biology  2013;426(3):645-655.
The catalytic moiety of Pseudomonas exotoxin A (domain III or PE3) inhibits protein synthesis by ADP-ribosylation of eukaryotic elongation factor 2 (eEF2). PE3 is widely used as a cytocidal payload in receptor-targeted protein toxin conjugates. We have designed and characterized catalytically inactive fragments of PE3 that are capable of structural complementation. We dissected PE3 at an extended loop and fused each fragment to one subunit of a heterospecific coiled coil. In vitro ADP-ribosylation and protein translation assays demonstrate that the resulting fusions — supplied exogenously as genetic elements or purified protein fragments — had no significant catalytic activity or effect on protein synthesis individually, but in combination catalyzed the ADP-ribosylation of eEF2 and inhibited protein synthesis. Although complementing PE3 fragments are less efficient catalytically than intact PE3 in cell-free systems, co-expression in live cells transfected with transgenes encoding the toxin fusions inhibits protein synthesis and causes cell death comparably as intact PE3. Complementation of split PE3 offers a direct extension of the immunotoxin approach to generate bispecific agents that may be useful to target complex phenotypes.
PMCID: PMC3997303  PMID: 24211469
exotoxin A; structural complementation; ADP-ribosylation; elongation factor 2; cytotoxicity
15.  Crystal structures of the first condensation domain of CDA synthetase suggest conformational changes during the synthetic cycle of nonribosomal peptide synthetases 
Journal of molecular biology  2013;425(17):3137-3150.
Nonribosomal peptide synthetases (NRPS) are large modular macromolecular machines that produce small peptide molecules with wide-ranging biological activities, such as antibiotics and green chemicals. The condensation (C) domain is responsible for amide bond formation, the central chemical step in nonribosomal peptide (NRP) synthesis. Here we present two crystal structures of the first condensation domain of the calcium-dependent antibiotic (CDA) synthetase (CDA-C1) from Streptomyces coelicolor, determined at resolutions of 1.8 Å and 2.4 Å. The conformations adopted by CDA-C1 are quite similar in these two structures, yet distinct from those seen in other NRPS C domain structures. High pressure liquid chromatography based reaction assays show that this CDA-C1 construct is catalytically active, and small angle X-ray scattering experiments suggest that the conformation observed in these crystal structures could faithfully represent the conformation in solution. We have performed targeted molecular dynamics simulations, normal mode analyses and energy minimized linear interpolation to investigate the conformational changes required to transition between the observed structures. We discuss the implications of these conformational changes in the synthetic cycle, and of the observation that the “latch” that covers the active site is consistently formed in all studied C domains.
PMCID: PMC4318696  PMID: 23756159 CAMSID: cams4143
16.  Distinct Features of Cap Binding by eIF4E1b Proteins 
Journal of Molecular Biology  2015;427(2):387-405.
eIF4E1b, closely related to the canonical translation initiation factor 4E (eIF4E1a), cap-binding protein is highly expressed in mouse, Xenopus and zebrafish oocytes. We have previously characterized eIF4E1b as a component of the CPEB mRNP translation repressor complex along with the eIF4E-binding protein 4E-Transporter, the Xp54/DDX6 RNA helicase and additional RNA-binding proteins. eIF4E1b exhibited only very weak interactions with m7GTP-Sepharose and, rather than binding eIF4G, interacted with 4E-T. Here we undertook a detailed examination of both Xenopus and human eIF4E1b interactions with cap analogues using fluorescence titration and homology modeling. The predicted structure of eIF4E1b maintains the α + β fold characteristic of eIF4E proteins and its cap-binding pocket is similarly arranged by critical amino acids: Trp56, Trp102, Glu103, Trp166, Arg112, Arg157 and Lys162 and residues of the C-terminal loop. However, we demonstrate that eIF4E1b is 3-fold less well able to bind the cap than eIF4E1a, both proteins being highly stimulated by methylation at N7 of guanine. Moreover, eIF4E1b proteins are distinguishable from eIF4E1a by a set of conserved amino acid substitutions, several of which are located near to cap-binding residues. Indeed, eIF4E1b possesses several distinct features, namely, enhancement of cap binding by a benzyl group at N7 position of guanine, a reduced response to increasing length of the phosphate chain and increased binding to a cap separated by a linker from Sepharose, suggesting differences in the arrangement of the protein's core. In agreement, mutagenesis of the amino acids differentiating eIF4E1b from eIF4E1a reduces cap binding by eIF4E1a 2-fold, demonstrating their role in modulating cap binding.
Graphical abstract
•Sequence analysis of vertebrate eIF4E1a and eIF4E1b proteins identified a set of conserved substitutions, including those near to cap-binding residues.•The fluorescence titration assay revealed that human and Xenopus eIF4E1b have 3-fold lower affinity for m7GTP than the eIF4E1a proteins.•Additional distinct features of cap binding by eIF4E1b suggest differences in the arrangement of the protein's core and its C-terminal loop.•Mutagenesis of the distinguishing amino acids reduced cap binding by eIF4E1a 2-fold, demonstrating their role in modulating affinity to m7GTP.
PMCID: PMC4306533  PMID: 25463438
eukaryotic initiation factor; eIF4E1b; translational repression; binding affinity; Xenopus laevis
17.  Interactions between the Leucine-zipper Motif of cGMP-Dependent Protein Kinase and the C-terminal Region of the Targeting Subunit of Myosin Light Chain Phosphatase* 
Journal of molecular biology  2007;373(5):1198-1212.
Nitric oxide induces vasodilation by elevating the production of cGMP, an activator of cGMP-dependent protein kinase (PKG). PKG subsequently causes smooth muscle relaxation in part via activation of myosin light chain phosphatase (MLCP). To date, the interaction between PKG and the targeting subunit of MLCP (MYPT1) is not fully understood. Earlier studies by one group of workers showed that the binding of PKG to MYPT1 is mediated by the leucine-zipper motifs at the N- and C-termini, respectively, of the two proteins. Another group, however, reported that binding of PKG to MYPT1 did not require the leucine-zipper motif of MYPT1. In this work we fully characterized the interaction between PKG and MYPT1 using biophysical techniques. For this purpose we constructed a recombinant PKG peptide corresponding to a predicted coiled coil region that contains the leucine-zipper motif. We further constructed various C-terminal MYPT1 peptides bearing various combinations of a predicted coiled coil region, extensions preceding this coiled coil region, and the leucine-zipper motif. Our results show, firstly, that while the leucine-zipper motif at the N-terminus of PKG forms a homodimeric coiled coil, the one at the C-terminus of MYPT1 is monomeric and non-helical. Secondly, the leucine-zipper motif of PKG binds to that of MYPT1 to form a heterodimer. Thirdly, when the leucine-zipper motif of MYPT1 is absent, the PKG leucine-zipper motif binds to the coiled coil region and upstream segments of MYPT1 via formation of a heterotetramer. These results provide rationalization of some of the findings by others using alternative binding analyses.
PMCID: PMC4310484  PMID: 17904578
smooth muscle; myosin phosphatase; protein kinase G; analytical ultracentrifugation; surface plasmon resonance
18.  Dimer-dimer interaction of the bacterial selenocysteine synthase SelA promotes functional active site formation and catalytic specificity 
Journal of molecular biology  2014;426(8):1723-1735.
The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins, and is synthesized on its specific tRNA (tRNASec). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNASec, formed by seryl-tRNA synthetase, to Sec-tRNASec. SelA, a member of the fold-type-I pyridoxal 5′-phosphate (PLP)-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500 kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNASec revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNASec. The SelA catalytic site is close to the dimer-dimer interface, but the significance of the dimer-pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions, and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of “depentamerized” SelA variants with mutations at the dimer-dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site, and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I PLP-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures.
PMCID: PMC4306587  PMID: 24456689
Fold-type-I PLP-dependent enzyme; selenium metabolism; tRNASec; pentamer of dimers; 21st amino acid
19.  Kinetic control in protein folding for light chain amyloidosis and the differential effects of somatic mutations 
Journal of molecular biology  2013;426(2):347-361.
Light chain amyloidosis is a devastating disease where immunoglobulin light chains form amyloid fibrils, resulting in organ dysfunction and death. Previous studies have shown a direct correlation between the protein thermodynamic stability and the propensity for amyloid formation for some proteins involved in light chain amyloidosis. Here we investigate the effect of somatic mutations on protein stability and in vitro fibril formation of single and double restorative mutants of the protein AL-103 compared to the wild type germline control protein. A scan rate dependence and hysteresis in the thermal unfolding and refolding was observed for all proteins. This indicates that the unfolding/refolding reaction is kinetically determined with different kinetic constants for unfolding and refolding even though the process remains experimentally reversible. Our structural analysis of AL-103 and AL-103 delP95aIns suggests a kinetic coupling of the unfolding/refolding process with cis-trans prolyl isomerization. Our data reveal that the deletion of Proline 95a (AL-103 delP95aIns), which removes the trans cis diproline motif present in the patient protein AL-103, results in a dramatic increment in the thermodynamic stability and a significant delay in fibril formation kinetics with respect to AL-103. Fibril formation is pH dependent; all proteins form fibrils at pH 2; reactions become slower and more stochastic as the pH increases up to pH 7. Based on these results, we propose that in addition to thermodynamic stability, kinetic stability (possibly influenced by the presence of cis Proline 95a), plays a major role in the AL-103 amyloid fibril formation process.
PMCID: PMC3892967  PMID: 24157440
20.  The Structure of Xis reveals the basis for Filament Formation and insight into DNA bending within a mycobacteriophage Intasome 
Journal of molecular biology  2013;426(2):412-422.
The Recombination Directionality Factor, Xis, is a DNA bending protein that determines the outcome of integrase-mediated site-specific recombination by redesign of higher-order protein-DNA architectures. Although the attachment site DNA of Mycobacteriophage Pukovnik is likely to contain four sites for Xis binding, Xis crystals contain five subunits in the asymmetric unit, four of which align into a Xis filament, and a fifth that is generated by an unusual domain swap. Extensive intersubunit contacts stabilize a bent filament-like arrangement with Xis monomers aligned head-to-tail. The structure implies a DNA bend of ~120°, which is in agreement with DNA bending measured in vitro. Formation of attR-containing intasomes requires only Int and Xis, distinguishing Pukovnik from lambda. Therefore, we conclude that in Pukovnik, Xis-induced DNA bending is sufficient to promote intramolecular Int-mediated bridges during intasome formation.
PMCID: PMC3902635  PMID: 24112940
DNA recombination; mycobacteriophage Pukovnik; Xis; DNA bending; filament; structure
21.  Cross-talk between diverse serine integrases 
Journal of molecular biology  2013;426(2):318-331.
Phage-encoded serine-integrases are large serine-recombinases that mediate integrative and excisive site-specific recombination of temperate phage genomes. They are well suited for use in heterologous systems and for synthetic genetic circuits as the attP and attB attachment sites are small (<50 bp), there are no host factor or DNA supercoiling requirements, and they are strongly directional, doing only excisive recombination in the presence of a recombination directionality factor. Combining different recombinases that function independently and without cross-talk to construct complex synthetic circuits is desirable, and several different serine-integrases are available. However, we show here that these functions are not reliably predictable, and we describe a pair of serine-integrases encoded by mycobacteriophages Bxz2 and Peaches with unusual and unpredictable specificities. The Integrases share only 59% amino acid sequence identity and the attP sites have fewer than 50% shared bases, but they use the same attB site and there is non-reciprocal cross-talk between the two systems. The DNA binding specificities do not result from differences in specific DNA contacts, but from the constraints imposed by the configuration of the component half-sites within each of the attachment site DNAs.
PMCID: PMC3947336  PMID: 24161951
22.  Positional effects of AAN motifs in rpoS regulation by sRNAs and Hfq 
Journal of molecular biology  2013;426(2):275-285.
The E. coli stationary phase transcription factor RpoS is translated in response to small noncoding RNAs (sRNAs), which base pair with the rpoS mRNA leader. The bacterial Sm-like protein Hfq anneals sRNAs with their mRNA targets by simultaneously binding the mRNA and sRNA. Intriguingly, Hfq is recruited to the rpoS leader via AAN motifs far upstream of the sRNA. SHAPE chemical footprinting showed that the rpoS leader is divided into a far upstream domain, an Hfq binding domain, and a downstream inhibitory stem-loop containing the sRNA and ribosome binding sites. To investigate how Hfq promotes sRNA-mRNA base pairing from a distance, the natural AAN Hfq binding site was deleted, and artificial AAN binding sites were inserted at various positions in the rpoS leader. All the relocated AAN motifs restored tight Hfq binding in vitro, but only insertion at the natural position restored Hfq-dependent sRNA annealing in vitro and sRNA regulation of rpoS translation in vivo. Furthermore, U-rich motifs in the downstream inhibitory domain stabilized the rpoS mRNA-Hfq complex and contributed to regulation of rpoS expression. We propose that the natural Hfq binding domain is optimal for positive regulation because it recruits Hfq to the mRNA and allows it to act on incoming sRNAs without opening the inhibitory stem-loop when sRNA are absent.
PMCID: PMC3947347  PMID: 24051417
RNA-protein interactions; bacterial stress response; translational control; RNA chaperone; 5′ UTR
23.  Binding of MgtR, a Salmonella Transmembrane Regulatory Peptide, to MgtC, a Mycobacterium tuberculosis Virulence Factor: a Structural Study 
Journal of molecular biology  2013;426(2):436-446.
MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium,inhibits growth in macrophages through binding to the membrane protein MgtC that has been identified as essential for replication in macrophages. While the Mycobacterium tuberculosis MgtC is highly homologuous to its S. Typhimurium analogue, there does not appear to be an Mtb homologue for MgtR, raising significant pharmacological interest in this system. Here, solid-state NMR and EPR spectroscopy in lipid bilayer preparations were used to demonstrate the formation of a heterodimer between S. Typhimurium MgtR and the transmembrane helix 4 of Mtb MgtC. Based on the experimental restraints, a structural model of this heterodimer was developed using computational techniques. The result is that MgtR appears to be ideally situated in the membrane to influence the functionality of MgtC.
PMCID: PMC3947350  PMID: 24140750
solid state nuclear magnetic resonance; electron spin resonance; restrained molecular dynamics; orientational restraints; distance restraints
24.  Locking the Active Conformation of c-Src Kinase through the Phosphorylation of the Activation Loop 
Journal of molecular biology  2013;426(2):423-435.
Molecular dynamics umbrella sampling simulations are used to compare the relative stability of the active conformation of the catalytic domain of c-Src kinase while the tyrosine 416 in the activation loop (A-loop) is either unphosphorylated or phosphorylated. When the A-loop is unphosphorylated, there is considerable flexiblity of the kinase. While the active conformation of the kinase is not forbidden and can be visited transiently, it is not the predominant state. This is consistent with the view that c-Src displays some catalytic activity even when the A-loop is unphosphorylated. In contrast, phosphorylation of the A-loop contributes to stabilize several structural features that are critical for catalysis, such as the hydrophobic regulatory spine, the HRD motif, and the electrostatic switch. In summary, the free energy landscape calculations demonstrate that phosphorylation of tyrosine 416 in the A-loop essentially “locks” the kinase into its catalytically competent conformation.
PMCID: PMC3947352  PMID: 24103328
25.  Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels 
Journal of molecular biology  2013;426(2):467-483.
Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail ‘neck’, are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the ‘outer ion’ site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies shows that this site forms a previously unknown determinant of CaV high affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily.
PMCID: PMC3947372  PMID: 24120938

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