PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-15 (15)
 

Clipboard (0)
None

Select a Filter Below

Journals
more »
Year of Publication
Document Types
1.  Mechanisms of HIV-1 Nef Function and Intracellular Signaling 
Advances in the last several years have enhanced mechanistic understanding of Nef induced CD4 and MHCI downregulation and have suggested a new paradigm for analyzing Nef function. In both of these cases, Nef acts by forming ternary complexes with significant contributions to stability imparted by non-canonical interactions. The mutational analyses and binding assays that have led to these conclusions are discussed. The recent progress has been dependent on conservative mutations and multi-protein binding assays. The poorly understood Nef functions of p21 activated protein kinase (PAK2) activation, enhancement of virion infectivity, and inhibition of immunoglobulin class switching are also likely to involve ternary complexes and non-canonical interactions. Hence, investigation of these latter Nef functions should benefit from a similar approach.
Six historically used alanine substitutions for determining structure-function relationships of Nef are discussed. These are M20A, E62A/E63A/E64A/E65A (AAAA), P72A/P75A (AXXA), R106A, L164A/L165A, and D174A/D175A. Investigations of less disruptive mutations in place of AAAA and AXXA have led to different interpretations of mechanism. Two recent examples of this alternate approach applied to PAK2 activation F191 and critical residue D123 are presented.
The implications of the new findings and the resulting new paradigm for Nef structure-function are discussed with respect to creating a map of Nef functions on the protein surface. We report the results of a PPI-Pred analysis for protein-protein interfaces. There are three predicted patches produced by the analysis which describe regions consistent with the currently known mutational analyses of Nef function.
doi:10.1007/s11481-011-9262-y
PMCID: PMC3777542  PMID: 21336563
HIV-1; Nef; non-canonical; CD4; MHCI; PAK2; infectivity; immunoglobulin class switching
2.  Evolution and Virulence Contributions of the Autotransporter Proteins YapJ and YapK of Yersinia pestis CO92 and Their Homologs in Y. pseudotuberculosis IP32953 
Infection and Immunity  2012;80(10):3693-3705.
Yersinia pestis, the causative agent of plague, evolved from the gastrointestinal pathogen Yersinia pseudotuberculosis. Both species have numerous type Va autotransporters, most of which appear to be highly conserved. In Y. pestis CO92, the autotransporter genes yapK and yapJ share a high level of sequence identity. By comparing yapK and yapJ to three homologous genes in Y. pseudotuberculosis IP32953 (YPTB0365, YPTB3285, and YPTB3286), we show that yapK is conserved in Y. pseudotuberculosis, while yapJ is unique to Y. pestis. All of these autotransporters exhibit >96% identity in the C terminus of the protein and identities ranging from 58 to 72% in their N termini. By extending this analysis to include homologous sequences from numerous Y. pestis and Y. pseudotuberculosis strains, we determined that these autotransporters cluster into a YapK (YPTB3285) class and a YapJ (YPTB3286) class. The YPTB3286-like gene of most Y. pestis strains appears to be inactivated, perhaps in favor of maintaining yapJ. Since autotransporters are important for virulence in many bacterial pathogens, including Y. pestis, any change in autotransporter content should be considered for its impact on virulence. Using established mouse models of Y. pestis infection, we demonstrated that despite the high level of sequence identity, yapK is distinct from yapJ in its contribution to disseminated Y. pestis infection. In addition, a mutant lacking both of these genes exhibits an additive attenuation, suggesting nonredundant roles for yapJ and yapK in systemic Y. pestis infection. However, the deletion of the homologous genes in Y. pseudotuberculosis does not seem to impact the virulence of this organism in orogastric or systemic infection models.
doi:10.1128/IAI.00529-12
PMCID: PMC3457547  PMID: 22802344
3.  The Crystal Structure of a Self-Activating G Protein α Subunit Reveals Its Distinct Mechanism of Signal Initiation 
Science signaling  2011;4(159):ra8.
In animals, heterotrimeric guanine nucleotide–binding protein (G protein) signaling is initiated by G protein–coupled receptors (GPCRs), which activate G protein α subunits; however, the plant Arabidopsis thaliana lacks canonical GPCRs, and its G protein α subunit (AtGPA1) is self-activating. To investigate how AtGPA1 becomes activated, we determined its crystal structure. AtGPA1 is structurally similar to animal G protein α subunits, but our crystallographic and biophysical studies revealed that it had distinct properties. Notably, the helical domain of AtGPA1 displayed pronounced intrinsic disorder and a tendency to disengage from the Ras domain of the protein. Domain substitution experiments showed that the helical domain of AtGPA1 was necessary for self-activation and sufficient to confer self-activation to an animal G protein α subunit. These findings reveal the structural basis for a mechanism for G protein activation in Arabidopsis that is distinct from the well-established mechanism found in animals.
doi:10.1126/scisignal.2001446
PMCID: PMC3551277  PMID: 21304159
4.  Heterotrimeric G-protein Signaling Is Critical to Pathogenic Processes in Entamoeba histolytica 
PLoS Pathogens  2012;8(11):e1003040.
Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGβγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism.
Author Summary
Entamoeba histolytica causes an estimated 50 million intestinal infections and 100,000 deaths per year worldwide. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, constituting a signaling pathway which, when perturbed, is seen to regulate multiple cellular processes required for pathogenesis. Like mammalian counterparts, EhGα1 forms a heterotrimer with EhGβγ that is dependent on guanine nucleotide exchange and hydrolysis. Despite engaging a classical G-protein effector, EhRGS-RhoGEF, EhGα1 diverges from mammalian Gα subunits and cannot be classified within mammalian Gα subfamilies, as highlighted by distinct structural features in our crystal structure of EhGα1 in the inactive conformation. To identify roles of G-protein signaling in pathogenesis-related cellular processes, we engineered trophozoites for inducible expression of EhGα1 or a dominant negative mutant, finding that G-protein signaling perturbation affects host cell attachment and the related process of contact-dependent killing, as well as trophozoite migration and Matrigel transmigration. A transcriptomic comparison of our engineered strains revealed differential expression of known virulence-associated genes, including amoebapores and cytotoxic cysteine proteases. The expression data suggested, and biochemical experiments confirmed, that cysteine protease secretion is altered upon G-protein overexpression, identifying a mechanism by which pathogenesis-related trophozoite behaviors are perturbed. In summary, E. histolytica encodes a vital heterotrimeric G-protein signaling pathway that is likely amenable to pharmacologic manipulation.
doi:10.1371/journal.ppat.1003040
PMCID: PMC3499586  PMID: 23166501
5.  AvrRpm1 Missense Mutations Weakly Activate RPS2-Mediated Immune Response in Arabidopsis thaliana 
PLoS ONE  2012;7(8):e42633.
Plants recognize microbes via specific pattern recognition receptors that are activated by microbe-associated molecular patterns (MAMPs), resulting in MAMP-triggered immunity (MTI). Successful pathogens bypass MTI in genetically diverse hosts via deployment of effectors (virulence factors) that inhibit MTI responses, leading to pathogen proliferation. Plant pathogenic bacteria like Pseudomonas syringae utilize a type III secretion system to deliver effectors into cells. These effectors can contribute to pathogen virulence or elicit disease resistance, depending upon the host plant genotype. In disease resistant genotypes, intracellular immune receptors, typically belonging to the nucleotide binding leucine-rich repeat family of proteins, perceive bacterial effector(s) and initiate downstream defense responses (effector triggered immunity) that include the hypersensitive response, and transcriptional re-programming leading to various cellular outputs that collectively halt pathogen growth. Nucleotide binding leucine-rich repeat sensors can be indirectly activated via perturbation of a host protein acting as an effector target. AvrRpm1 is a P. syringae type III effector. Upon secretion into the host cell, AvrRpm1 is acylated by host enzymes and directed to the plasma membrane, where it contributes to virulence. This is correlated with phosphorylation of Arabidopsis RIN4 in vivo. RIN4 is a negative regulator of MAMP-triggered immunity, and its modification in the presence of four diverse type III effectors, including AvrRpm1, likely enhances this RIN4 regulatory function. The RPM1 nucleotide binding leucine-rich repeat sensor perceives RIN4 perturbation in disease resistant plants, leading to a successful immune response. Here, demonstrate that AvrRpm1 has a fold homologous to the catalytic domain of poly(ADP-ribosyl) polymerase. Site-directed mutagenesis of each residue in the putative catalytic triad, His63-Tyr122-Asp185 of AvrRpm1, results in loss of both AvrRpm1-dependent virulence and AvrRpm1-mediated activation of RPM1, but, surprisingly, causes a gain of function: the ability to activate the RPS2 nucleotide binding leucine-rich repeat sensor.
doi:10.1371/journal.pone.0042633
PMCID: PMC3412798  PMID: 22880057
6.  N-terminal strands of filamin Ig domains act as a conformational switch under biological forces 
Proteins  2010;78(1):12-24.
Conformational changes of filamin A under stress have been postulated to play crucial roles in signaling pathways of cell responses. Direct observation of conformational changes under stress is beyond the resolution of current experimental techniques. On the other hand, computational studies are mainly limited to either traditional molecular dynamics simulations of short durations and high forces or simulations of simplified models. Here we perform all-atom discrete molecular dynamics (DMD) simulations to study thermally and force-induced unfolding of filamin A. The high conformational sampling efficiency of DMD allows us to observe force-induced unfolding of filamin A Ig domains under physiological forces. The computationally identified critical unfolding forces agree well with experimental measurements. Despite a large heterogeneity in the population of force-induced intermediate states, we find a common initial unfolding intermediate in all the Ig domains of filamin, where the N-terminal strand unfolds. We also study the thermal unfolding of several filamin Ig-like domains. We find that thermally induced unfolding features an early-stage intermediate state similar to the one observed in force-induced unfolding and characterized by N-terminal strand being unfurled. We propose that the N-terminal strand may act as a conformational switch that unfolds under physiological forces leading to exposure of cryptic binding sites, removal of native binding sites, and modulating the quaternary structure of domains.
doi:10.1002/prot.22479
PMCID: PMC2804786  PMID: 19514078
7.  Evolution of a Signaling Nexus Constrained by Protein Interfaces and Conformational States 
PLoS Computational Biology  2010;6(10):e1000962.
Heterotrimeric G proteins act as the physical nexus between numerous receptors that respond to extracellular signals and proteins that drive the cytoplasmic response. The Gα subunit of the G protein, in particular, is highly constrained due to its many interactions with proteins that control or react to its conformational state. Various organisms contain differing sets of Gα-interacting proteins, clearly indicating that shifts in sequence and associated Gα functionality were acquired over time. These numerous interactions constrained much of Gα evolution; yet Gα has diversified, through poorly understood processes, into several functionally specialized classes, each with a unique set of interacting proteins. Applying a synthetic sequence-based approach to mammalian Gα subunits, we established a set of seventy-five evolutionarily important class-distinctive residues, sites where a single Gα class is differentiated from the three other classes. We tested the hypothesis that shifts at these sites are important for class-specific functionality. Importantly, we mapped known and well-studied class-specific functionalities from all four mammalian classes to sixteen of our class-distinctive sites, validating the hypothesis. Our results show how unique functionality can evolve through the recruitment of residues that were ancestrally functional. We also studied acquisition of functionalities by following these evolutionarily important sites in non-mammalian organisms. Our results suggest that many class-distinctive sites were established early on in eukaryotic diversification and were critical for the establishment of new Gα classes, whereas others arose in punctuated bursts throughout metazoan evolution. These Gα class-distinctive residues are rational targets for future structural and functional studies.
Author Summary
Proteins evolve new protein-protein interactions through changes to their residues. Many residue changes are harmful because they disrupt important existing interactions and functions. The more interactions a protein participates in, the more difficult it is to make changes that are not harmful to the protein. And yet, proteins with many existing interactions are also likely to evolve new functions or new interactions. How does evolution occur in the context of a well-constrained protein with many interactions? We studied the heterotrimeric G protein subunit Gα, a multi-functional protein that acts at the nexus between receptors responding to extracellular signals and the cytoplasmic proteins driving the response within the cell. The Gα subunit participates in numerous interactions that have constrained much of Gα evolution; yet Gα has diversified into four functionally specialized classes. We developed an approach that identifies key residue changes important to the evolution of Gα functionality and class, and gained insight into the types of residue changes that occurred both early and late in the evolution of Gα function. By studying these critical residues in Gα we can de-couple the many functionalities of this signaling nexus.
doi:10.1371/journal.pcbi.1000962
PMCID: PMC2954821  PMID: 20976244
8.  Prediction of Protein-Protein Interfaces on G-Protein β Subunits Reveals a Novel Phospholipase C β2 Binding Domain 
Journal of molecular biology  2009;392(4):1044-1054.
Gβ subunits from heterotrimeric G-proteins directly bind diverse proteins, including effectors and regulators, to modulate a wide array of signaling cascades. These numerous interactions constrained the evolution of the molecular surface of Gβ. Though mammals contain five Gβ genes comprising two classes (Gβ1-like and Gβ5-like), plants and fungi have a single ortholog and organisms such as Caenorhabditis elegans and Drosophila melanogaster contain one copy from each class. A limited number of crystal structures of complexes containing Gβ subunits and complementary biochemical data highlight specific sites within Gβs needed for protein interactions. It is difficult to determine from these interaction sites what, if any, additional regions of the Gβ molecular surface comprise interaction interfaces essential to Gβ's role as a nexus in numerous signaling cascades. We used a comparative evolutionary approach to identify five known and eight previously-unknown putative interfaces on the surface of Gβ. We show that one such novel interface occurs between Gβ and phospholipase C β2 (PLC-β2), a mammalian Gβ interacting protein. Substitutions of residues within this Gβ-PLC-β2 interface reduce the activation of PLC-β2 by Gβ1, confirming that our de novo comparative evolutionary approach predicts previously unknown Gβ-protein interfaces. Similarly, we hypothesize the seven remaining untested novel regions contribute to putative interfaces for other Gβ interacting proteins. Finally, this comparative evolutionary approach is suitable for application to any protein involved in a significant number of protein-protein interactions.
doi:10.1016/j.jmb.2009.07.076
PMCID: PMC2767172  PMID: 19646992
Heterotrimeric G proteins; Phospholipase C-β2; Protein surface evolution; Interface prediction; PLC-β2 – Gβγ interaction interface
9.  X-ray crystal structure of the rotavirus inner capsid particle at 3.8 Å resolution 
Journal of molecular biology  2010;397(2):587-599.
The rotavirus inner capsid particle, known as the “double-layered particle” (DLP), is the “payload” delivered into a cell in the process of viral infection. Its inner and outer protein layers, composed of VP2 and VP6, respectively, package the eleven segments of double-stranded RNA (dsRNA) of the viral genome, as well as about the same number of polymerase molecules (VP1) and capping-enzyme molecules (VP3). We have determined the crystal structure of the bovine rotavirus DLP. There is one full particle (outer diameter ~700 Å) in the asymmetric unit of the P212121 unit cell, of dimensions a= 740 Å, b= 1198 Å, c= 1345 Å. A three-dimensional reconstruction from electron cryomicroscopy was used as a molecular-replacement model for initial phase determination to about 18.5 Å resolution and the sixty-fold redundancy of the icosahedral particle symmetry allowed phases to be extended stepwise to the limiting resolution of the data (3.8 Å). The structure of a VP6 trimer (determined previously by others) fits the outer-layer density with very little adjustment. The T=13 triangulation number of that layer implies that there are four and one-third VP6 trimers per icosahedral asymmetric unit. The inner layer has 120 copies of VP2 and thus two per icosahedral asymmetric unit, designated VP2A and VP2B. Residues 101-880 fold into a relatively thin, principal domain, comma-like in outline, shaped such that only rather modest distortions (concentrated at two “subdomain” boundaries) allow VP2A and B to form a uniform layer with essentially no gaps at the subunit boundaries, except for a modest pore along the fivefold axis. The VP2 principal domain resembles those of the corresponding shells and homologous proteins in other dsRNA viruses: λ1 in orthoreoreoviruses, VP3 in orbiviruses. Residues 1-80 of VP2A and VP2B fold together with four other such pairs into a “fivefold hub” that projects into the DLP interior along the fivefold axis; residues 81-100 link the ten polypeptide chains emerging from a fivefold hub to the N-termini of their corresponding principal domains, clustered into a decameric assembly unit. The fivefold hub appears to have several distinct functions. One is to recruit a copy of VP1 (or of a VP1–VP3 complex), potentially along with a segment of (+)-strand RNA, as a decamer of VP2 assembles. A second is to serve as a shaft around which can coil a segment of dsRNA. A third is to guide nascent mRNA, synthesized in the DLP interior by VP1 and 5′-capped by the action of VP3, out through a fivefold exit channel. We propose a model for rotavirus particle assembly, based on known requirements for virion formation together with the structure of the DLP and that of VP1, determined earlier.
doi:10.1016/j.jmb.2010.01.055
PMCID: PMC2860780  PMID: 20122940
double-layered particle; double-stranded RNA; virus assembly; icosahedral symmetry
10.  Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway† 
Biochemistry  2009;48(27):6390-6401.
G protein-coupled receptors mediate cellular responses to a wide variety of stimuli, including taste, light and neurotransmitters. In the yeast Saccharomyces cerevisiae, activation of the pheromone pathway triggers events leading to mating. The view had long been held that the G protein-mediated signal occurs principally at the plasma membrane. Recently, it has been shown that the G protein α subunit Gpa1 can promote signaling at endosomes and requires two components of the sole phosphatidylinositol-3-kinase in yeast, Vps15 and Vps34. Vps15 contains multiple WD repeats and also binds to Gpa1 preferentially in the GDP-bound state; these observations led us to hypothesize that Vps15 may function as a G protein β subunit at the endosome. Here we show an X-ray crystal structure of the Vps15 WD domain that reveals a seven-bladed propeller resembling that of typical Gβ subunits. We show further that the WD domain is sufficient to bind Gpa1 as well as to Atg14, a potential Gγ protein that exists in a complex with Vps15. The Vps15 kinase domain together with the intermediate domain (linking the kinase and WD domains) also contributes to Gpa1 binding, and is necessary for Vps15 to sustain G protein signaling. These findings reveal that the Vps15 Gβ-like domain serves as a scaffold to assemble Gpa1 and Atg14, whereas the kinase and intermediate domains are required for proper signaling at the endosome.
doi:10.1021/bi900621w
PMCID: PMC2740480  PMID: 19445518
11.  G Protein–Coupled Receptor Sorting to Endosomes and Lysosomes 
The heptahelical G protein–coupled receptors (GPCRs) belong to the largest family of cell surface signaling receptors encoded in the human genome. GPCRs signal to diverse extracellular stimuli and control a vast number of physiological responses, making this receptor class the target of nearly half the drugs currently in use. In addition to rapid desensitization, receptor trafficking is crucial for the temporal and spatial control of GPCR signaling. Sorting signals present in the intracytosolic domains of GPCRs regulate trafficking through the endosomal-lysosomal system. GPCR internalization is mediated by serine and threonine phosphorylation and arrestin binding. Short, linear peptide sequences including tyrosine- and dileucine-based motifs, and PDZ ligands that are recognized by distinct endocytic adaptor proteins also mediate internalization and endosomal sorting of GPCRs. We present new data from bioinformatic searches that reveal the presence of these types of sorting signals in the cytoplasmic tails of many known GPCRs. Several recent studies also indicate that the covalent modification of GPCRs with ubiquitin serves as a signal for internalization and lysosomal sorting, expanding the diversity of mechanisms that control trafficking of mammalian GPCRs.
doi:10.1146/annurev.pharmtox.48.113006.094646
PMCID: PMC2869288  PMID: 17995450
GPCR; arrestin; ubiquitin; trafficking; clathrin; PDZ; bioinformatic
12.  Characterization of calcium- and integrin-binding protein 1 (CIB1) knockout platelets: Potential compensation by CIB family members 
Thrombosis and haemostasis  2008;100(5):847-856.
Summary
Platelet aggregation requires activation of the αIIbβ3 integrin, an event regulated by the integrin cytoplasmic tails. CIB1 binds to the cytoplasmic tail of the integrin αIIb subunit. Previous overexpression and knockdown studies in murine megakaryocytes demonstrated that CIB1 inhibits integrin αIIbβ3 activation. Here we analyzed Cib1−/− mice to determine the function of CIB1 in platelets in vitro and in vivo. We found that although these mice had no overt platelet phenotype, mRNA level of CIB1 homolog CIB3 was increased in Cib1−/− megakaryocytes. In vitro binding experiments showed that recombinant CIB1, -2 and -3 bound specifically to an αIIb cytoplasmic tail peptide. Subsequent protein modeling experiments indicated that CIBs 1–3 each have a highly conserved hydrophobic binding pocket. Therefore, the potential exists for compensation for the loss of CIB1 by these CIB family members, thereby preventing pathologic thrombus formation in Cib1−/− mice.
PMCID: PMC2771430  PMID: 18989529
CIB1; platelet; integrin αIIbβ3 activation; aggregation; knockout mouse; compensation
13.  Structural basis for the sequence-dependent effects of platinum–DNA adducts 
Nucleic Acids Research  2009;37(8):2434-2448.
The differences in efficacy and molecular mechanisms of platinum based anti-cancer drugs cisplatin (CP) and oxaliplatin (OX) have been hypothesized to be in part due to the differential binding affinity of cellular and damage recognition proteins to CP and OX adducts formed on adjacent guanines in genomic DNA. HMGB1a in particular exhibits higher binding affinity to CP-GG adducts, and the extent of discrimination between CP- and OX-GG adducts is dependent on the bases flanking the adducts. However, the structural basis for this differential binding is not known. Here, we show that the conformational dynamics of CP- and OX-GG adducts are distinct and depend on the sequence context of the adduct. Molecular dynamics simulations of the Pt-GG adducts in the TGGA sequence context revealed that even though the major conformations of CP- and OX-GG adducts were similar, the minor conformations were distinct. Using the pattern of hydrogen bond formation between the Pt–ammines and the adjacent DNA bases, we identified the major and minor conformations sampled by Pt–DNA. We found that the minor conformations sampled exclusively by the CP-GG adduct exhibit structural properties that favor binding by HMGB1a, which may explain its higher binding affinity to CP-GG adducts, while these conformations are not sampled by OX-GG adducts because of the constraints imposed by its cyclohexane ring, which may explain the negligible binding affinity of HMGB1a for OX-GG adducts in the TGGA sequence context. Based on these results, we postulate that the constraints imposed by the cyclohexane ring of OX affect the DNA conformations explored by OX-GG adduct compared to those of CP-GG adduct, which may influence the binding affinities of HMG-domain proteins for Pt-GG adducts, and that these conformations are further influenced by the DNA sequence context of the Pt-GG adduct.
doi:10.1093/nar/gkp029
PMCID: PMC2677858  PMID: 19255091
14.  Active Nuclear Receptors Exhibit Highly Correlated AF-2 Domain Motions 
PLoS Computational Biology  2008;4(7):e1000111.
Nuclear receptor ligand binding domains (LBDs) convert ligand binding events into changes in gene expression by recruiting transcriptional coregulators to a conserved activation function-2 (AF-2) surface. While most nuclear receptor LBDs form homo- or heterodimers, the human nuclear receptor pregnane X receptor (PXR) forms a unique and essential homodimer and is proposed to assemble into a functional heterotetramer with the retinoid X receptor (RXR). How the homodimer interface, which is located 30 Å from the AF-2, would affect function at this critical surface has remained unclear. By using 20- to 30-ns molecular dynamics simulations on PXR in various oligomerization states, we observed a remarkably high degree of correlated motion in the PXR–RXR heterotetramer, most notably in the four helices that create the AF-2 domain. The function of such correlation may be to create “active-capable” receptor complexes that are ready to bind to transcriptional coactivators. Indeed, we found in additional simulations that active-capable receptor complexes involving other orphan or steroid nuclear receptors also exhibit highly correlated AF-2 domain motions. We further propose a mechanism for the transmission of long-range motions through the nuclear receptor LBD to the AF-2 surface. Taken together, our findings indicate that long-range motions within the LBD scaffold are critical to nuclear receptor function by promoting a mobile AF-2 state ready to bind coactivators.
Author Summary
Long-range motions play essential roles in protein function but are difficult to appreciate from static crystal structures. We sought to understand how macromolecular motion affects the formation of transcriptional complexes central to controlling gene expression. Using 20- to 30-ns molecular dynamics simulations, we examined three nuclear receptors that function as ligand-regulated transcription factors: the pregnane X receptor, the peroxisome proliferator-activator receptor-γ, and estrogen receptor-α. We found that each of these receptors exhibits a high degree of correlated motions within the domain responsible for forming functionally essential protein–protein interactions with transcriptional coactivators. We further found that specific long-range (up to 30 Å) motions play an important role in these dynamics. Our results show that “active-capable” nuclear receptors are prepared for coactivator contacts by maintaining a mobile but preformed protein–protein interaction surface.
doi:10.1371/journal.pcbi.1000111
PMCID: PMC2432469  PMID: 18617990
15.  Conformational Dynamics of the Major Yeast Phosphatidylinositol Transfer Protein Sec14p: Insight into the Mechanisms of Phospholipid Exchange and Diseases of Sec14p-Like Protein Deficiencies 
Molecular Biology of the Cell  2007;18(5):1928-1942.
Molecular dynamics simulations coupled with functional analyses of the major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p identify structural elements involved in regulating the ability of Sec14p to execute phospholipid exchange. The molecular dynamics simulations suggest large rigid body motions within the Sec14p molecule accompany closing and opening of an A10/T4/A11 helical gate, and that “state-of-closure” of this helical gate determines access to the Sec14p phospholipid binding cavity. The data also project that conformational dynamics of the helical gate are controlled by a hinge unit (residues F212, Y213, K239, I240, and I242) that links to the N- and C-terminal ends of the helical gate, and by a novel gating module (composed of the B1LB2 and A12LT5 substructures) through which conformational information is transduced to the hinge. The 114TDKDGR119 motif of B1LB2 plays an important role in that transduction process. These simulations offer new mechanistic possibilities for an important half-reaction of the Sec14p phospholipid exchange cycle that occurs on membrane surfaces after Sec14p has ejected bound ligand, and is reloading with another phospholipid molecule. These conformational transitions further suggest structural rationales for known disease missense mutations that functionally compromise mammalian members of the Sec14-protein superfamily.
doi:10.1091/mbc.E06-11-1024
PMCID: PMC1855008  PMID: 17344474

Results 1-15 (15)