Structural insight into GRIP1-PDZ6 in Alzheimer’s disease: study from protein expression data to molecular dynamics simulations

Protein–protein interaction domain, PDZ, plays a critical role in efficient synaptic transmission in brain. Dysfunction of synaptic transmission is thought to be the underlying basis of many neuropsychiatric and neurodegenerative disorders including Alzheimer’s disease (AD). In this study, Glutamate Receptor Interacting Protein1 (GRIP1) was identified as one of the most important differentially expressed, topologically significant proteins in the protein–protein interaction network. To date, very few studies have analyzed the detailed structural basis of PDZ-mediated protein interaction of GRIP1. In order to gain better understanding of structural and dynamic basis of these interactions, we employed molecular dynamics (MD) simulations of GRIP1-PDZ6 dimer bound with Liprin-alpha and GRIP1-PDZ6 dimer alone each with 100 ns simulations. The analyses of MD simulations of Liprin-alpha bound GRIP1-PDZ6 dimer show considerable conformational differences than that of peptide-free dimer in terms of SASA, hydrogen bonding patterns, and along principal component 1 (PC1). Our study also furnishes insight into the structural attunement of the PDZ6 domains of Liprin-alpha bound GRIP1 that is attributed by significant shift of the Liprin-alpha recognition helix in the simulated peptide-bound dimer compared to the crystal structure and simulated peptide-free dimer. It is evident that PDZ6 domains of peptide-bound dimer show differential movements along PC1 than that of peptide-free dimers. Thus, Liprin-alpha also serves an important role in conferring conformational changes along the dimeric interface of the peptide-bound dimer. Results reported here provide information that may lead to novel therapeutic approaches in AD.     

The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization

The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form.     

Residues in the HIV-1 capsid assembly inhibitor binding site are essential for maintaining the assembly-competent quaternary structure of the capsid protein

Morphogenesis of infectious HIV-1 involves budding of immature virions followed by proteolytic disassembly of the Gag protein shell and subsequent assembly of processed capsid proteins (CA) into the mature HIV-1 core. The dimeric interface between C-terminal domains of CA (C-CA) has been shown to be important for both immature and mature assemblies. We previously reported a CA-binding peptide (CAI) that blocks both assembly steps in vitro. The three-dimensional structure of the C-CA/CAI complex revealed an allosteric effect of CAI that alters the C-CA dimer interface. Based on this structure, we now investigated the phenotypes of mutations in the binding pocket. CA variants carrying mutations Y169A, L211A, or L211S had a reduced affinity for CAI and were unable to form mature-like particles in vitro. These mutations also blocked morphological conversion to mature virions in tissue culture and abolished infectivity. X-ray crystallographic analyses of the variant C-CA domains revealed that these alterations induced the same allosteric change at the dimer interface observed in the C-CA/CAI complex. These results point to a role of key interactions between conserved amino acids in the CAI binding pocket of C-CA in maintaining the correct conformation necessary for mature core assembly.     

Long α helices projecting from the membrane as the dimer interface in the voltage-gated H+ channel

The voltage-gated H⁺ channel (Hv) is a H⁺-permeable voltage-sensor domain (VSD) protein that consists of four transmembrane segments (S1–S4). Hv assembles as a dimeric channel and two transmembrane channel domains function cooperatively, which is mediated by the coiled-coil assembly domain in the cytoplasmic C terminus. However, the structural basis of the interdomain interactions remains unknown. Here, we provide a picture of the dimer configuration based on the analyses of interactions among two VSDs and a coiled-coil domain. Systematic mutations of the linker region between S4 of VSD and the coiled-coil showed that the channel gating was altered in the helical periodicity with the linker length, suggesting that two domains are linked by helices. Cross-linking analyses revealed that the two S4 helices were situated closely in the dimeric channel. The interaction interface between the two S4 and the assembly interface of the coiled-coil domain were aligned in the same direction based on the phase angle calculation along α helices. Collectively, we propose that continuous helices stretching from the transmembrane to the cytoplasmic region in the dimeric interface regulate the channel activation in the Hv dimer.     

Dynamic features of homodimer interfaces calculated by normal‐mode analysis

Knowledge of the dynamic features of protein interfaces is necessary for a deeper understanding of protein–protein interactions. We performed normal‐mode analysis (NMA) of 517 nonredundant homodimers and their protomers to characterize dimer interfaces from a dynamic perspective. The motion vector calculated by NMA for each atom of a dimer was decomposed into internal and external motion vectors in individual component subunits, followed by the averaging of time‐averaged correlations between these vectors over atom pairs in the interface. This averaged correlation coefficient (ACC) was defined for various combinations of vectors and investigated in detail. ACCs decrease exponentially with an increasing interface area and r‐value, that is, interface area divided by the entire subunit surface area. As the r‐value reflects the nature of dimer formation, the result suggests that both the interface area and the nature of dimer formation are responsible for the dynamic properties of dimer interfaces. For interfaces with small or medium r‐values and without intersubunit entanglements, ACCs are found to increase on dimer formation when compared with those in the protomer state. In contrast, ACCs do not increase on dimer formation for interfaces with large r‐values and intersubunit entanglements such as in interwinding dimers. Furthermore, relationships between ACCs for intrasubunit atom pairs and for intersubunit atom pairs are found to significantly differ between interwinding and noninterwinding dimers for external motions. External motions are considered as an important factor for characterizing dimer interfaces.     

An isoform-selective inhibitor of tropomyosin receptor kinase A behaves as molecular glue

The production of TrkA-selective inhibitors is considerably difficult because the kinase domains of TrkA and its isoforms TrkB/C have highly homologous amino acid sequences. Here we describe the structural basis for the acquisition of selectivity for a isoform-selective TrkA inhibitor, namely compound V1. The X-ray structure revealed that V1 acts as a molecular glue to stabilize the symmetrical dimer of the TrkA kinase domains. V1 binds to the ATP-binding site and simultaneously engages in the dimeric interface of TrkA. The region of the dimeric interface in TrkA is not conserved in TrkB/C; thus, dimer formation may be a novel mechanism for the production of selective TrkA inhibitors. The biochemical and biophysical assay results confirmed that V1 selectively inhibited TrkA and induced the dimer formation of TrkA, but not TrkB. The binding pocket at the TrkA dimer interface can be used for the production of new isoform-selective TrkA inhibitors.     

Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: a model for changes in dynamics upon target binding

Low molecular weight protein tyrosine phosphatase (LMW-PTP) dimerizes in the phosphate-bound state in solution with a dissociation constant of K(d)=1.5(+/-0.1)mM and an off-rate on the order of 10(4)s(-1). 1H and 15N NMR chemical shifts identify the dimer interface, which is in excellent agreement with that observed in the crystal structure of the dimeric S19A mutant. Two tyrosine residues of each molecule interact with the active site of the other molecule, implying that the dimer may be taken as a model for a complex between LMW-PTP and a target protein. 15N relaxation rates for the monomeric and dimeric states were extrapolated from relaxation data acquired at four different protein concentrations. Relaxation data of satisfactory precision were extracted for the monomer, enabling model-free analyses of backbone fluctuations on pico- to nanosecond time scales. The dimer relaxation data are of lower quality due to extrapolation errors and the possible presence of higher-order oligomers at higher concentrations. A qualitative comparison of order parameters in the monomeric and apparent dimeric states shows that loops forming the dimer interface become rigidified upon dimerization. Qualitative information on monomer-dimer exchange and intramolecular conformational exchange was obtained from the concentration dependence of auto- and cross-correlated relaxation rates. The loop containing the catalytically important Asp129 fluctuates between different conformations in both the monomeric and dimeric (target bound) states. The exchange rate compares rather well with that of the catalyzed reaction step, supporting existing hypotheses that catalysis and enzyme dynamics may be coupled. The side-chain of Trp49, which is important for substrate specificity, exhibits conformational dynamics in the monomer that are largely quenched upon formation of the dimer, suggesting that binding is associated with the selection of a single side-chain conformer.     

Molecular basis for the structural instability of human DJ-1 induced by the L166P mutation associated with Parkinson's disease

DJ-1 is a dimeric protein of unknown function in vivo. A mutation in the human DJ-1 gene causing substitution of proline for leucine at residue 166 (L166P) has been linked to early onset Parkinson's disease. Lack of structural stability has precluded experimental determination of atomic-resolution structures of the L166P DJ-1 polymorph. We have performed multiple molecular dynamics (MD) simulations ( approximately 1/3 mus) of the wild-type and L166P DJ-1 polymorph at physiological temperature to predict specific structural effects of the L166P substitution. L166P disrupted helices alpha1, alpha5, alpha6 and alpha8 with alpha8 undergoing particularly severe disruption. Secondary structural elements critical for protein stability and dimerization were significantly disrupted across the entire dimer interface, as were extended hydrophobic surfaces involved in dimer formation. Relative to wild-type DJ-1, L166P DJ-1 populated a broader ensemble of structures, many of which corresponded to distorted conformations. In a L166P dimer model the substitution significantly destabilized the dimer interface, interrupting >100 intermolecular contacts that are important for dimer formation. The L166P substitution also led to major perturbations in the region of a highly conserved cysteine residue (Cys-106) that participates in dimerization and that is critical for a proposed chaperone function of DJ-1. Cys-106 is located approximately 16 A from the substitution site, demonstrating that structural disruptions propagate throughout the whole protein. Furthermore, L166P DJ-1 showed a significant increase in hydrophobic surface area relative to wild-type protein, possibly explaining the tendency of the mutant protein to aggregate. These simulations provide details about specific structural disturbances throughout L166P DJ-1 that previous studies have not revealed.     

Insights into dimerization and four-helix bundle formation found by dissection of the dimer interface of the GrpE protein from Escherichia coli

The GrpE heat shock protein from Escherichia coli has a homodimeric structure. The dimer interface encompasses two long alpha-helices at the NH(2)-terminal end from each monomer (forming a "tail"), which lead into a small four-helix bundle from which each monomer contributes two short sequential alpha-helices in an antiparallel topological arrangement. We have created a number of different deletion mutants of GrpE that have portions of the dimer interface to investigate requirements for dimerization and to study four-helix bundle formation. Using chemical crosslinking and analytical ultracentrifugation techniques to probe for multimeric states, we find that a mutant containing only the long alpha-helical tail portion (GrpE1-88) is unable to form a dimer, most likely due to a decrease in alpha-helical content as determined by circular dichroism spectroscopy, thus one reason for a dimeric structure for the GrpE protein is to support the tail region. Mutants containing both of the short alpha-helices (GrpE1-138 and GrpE88-197) are able to form a dimer and presumably the four-helix bundle at the dimer interface. These two mutants have equilibrium constants for the monomer-dimer equilibrium that are very similar to the full-length protein suggesting that the tail region does not contribute significantly to the stability of the dimer. Interestingly, one mutant that contains just one of the short alpha-helices (GrpE1-112) exists as a tetrameric species, which presumably is forming a four-helix bundle structure. A proposed model is discussed for this mutant and its relevance for factors influencing four-helix bundle formation.     

A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle

The ATPase components of ATP binding cassette (ABC) transporters power the transporters by binding and hydrolyzing ATP. Major conformational changes of an ATPase are revealed by crystal structures of MalK, the ATPase subunit of the maltose transporter from Escherichia coli, in three different dimeric configurations. While other nucleotide binding domains or subunits display low affinity for each other in the absence of the transmembrane segments, the MalK dimer is stabilized through interactions of the additional C-terminal domains. In the two nucleotide-free structures, the N-terminal nucleotide binding domains are separated to differing degrees, and the dimer is maintained through contacts of the C-terminal regulatory domains. In the ATP-bound form, the nucleotide binding domains make contact and two ATPs lie buried along the dimer interface. The two nucleotide binding domains of the dimer open and close like a pair of tweezers, suggesting a regulatory mechanism for ATPase activity that may be tightly coupled to translocation.     

Specificity of helix packing in transmembrane dimer of the cell death factor BNIP3: a molecular modeling study

BNIP3 is a mitochondrial 19-kDa proapoptotic protein, a member of the Bcl-2 family. It has a single COOH-terminal transmembrane (TM) alpha-helical domain, which is required for membrane targeting, proapoptotic activity, hetero- and homo-dimerization in membrane. The role and the molecular details of association of TM helices of BNIP3 are yet to be established. Here, we present a molecular modeling study of helix interactions in its membrane domain. The approach combines Monte Carlo conformational search in an implicit hydrophobic slab followed by molecular dynamics simulations in a hydrated full-atom lipid bilayer. The former technique was used for exhaustive sampling of the peptides' conformational space and for generation of putative "native-like" structures of the dimer. The latter ones were taken as realistic starting points to assess stability and dynamic behavior of the complex in explicit lipid-water surrounding. As a result, several groups of tightly packed right-handed structures of the dimer were proposed. They have almost similar helix-helix interface, which includes the motif A(176)xxxG(180)xxxG(184) and agrees well with previous mutagenesis data and preliminary NMR analysis. Molecular dynamics simulations of these structures reveal perfect adaptation of most of them to heterogeneous membrane environment. A remarkable feature of the predicted dimeric structures is the occurrence of a cluster of H-bonded histidine 173 and serines 168 and 172 on the helix interface, near the N-terminus. Because of specific polar interactions between the monomers, this part of the dimer has no such dense packing as the C-terminal one, thus allowing penetration of water from the extramembrane side into the membrane interior. We propose that the ionization state of His(173) can mediate structural and dynamic properties of the dimer. This, in turn, may be related to pH-dependent proapoptotic activity of BNIP3, which is triggering on by acidosis appearing under hypoxic conditions.     

Monomeric state of S100P protein: Experimental and molecular dynamics study

S100 proteins constitute a large subfamily of the EF-hand superfamily of calcium binding proteins. They possess one classical EF-hand Ca²⁺-binding domain and an atypical EF-hand domain. Most of the S100 proteins form stable symmetric homodimers. An analysis of literature data on S100 proteins showed that their physiological concentrations could be much lower than dissociation constants of their dimeric forms. It means that just monomeric forms of these proteins are important for their functioning. In the present work, thermal denaturation of apo-S100P protein monitored by intrinsic tyrosine fluorescence has been studied at various protein concentrations within the region from 0.04–10 μM. A transition from the dimeric to monomeric form results in a decrease in protein thermal stability shifting the mid-transition temperature from 85 to 75 °C. Monomeric S100P immobilized on the surface of a sensor chip of a surface plasmon resonance instrument forms calcium dependent 1 to 1 complexes with human interleukin-11 (equilibrium dissociation constant 1.2 nM). In contrast, immobilized interleukin-11 binds two molecules of dimeric S100P with dissociation constants of 32 nM and 288 nM. Since effective dissociation constant of dimeric S100P protein is very low (0.5 μM as evaluated from our data) the sensitivity of the existing physical methods does not allow carrying out a detailed study of S100P monomer properties. For this reason, we have used molecular dynamics methods to evaluate structural changes in S100P upon its transition from the dimeric to monomeric state. 80-ns molecular dynamics simulations of kinetics of formation of S100P, S100B and S100A11 monomers from the corresponding dimers have been carried out. It was found that during the transition from the homo-dimer to monomer form, the three S100 monomer structures undergo the following changes: (1) the helices in the four-helix bundles within each monomer rotate in order to shield the exposed non-polar residues; (2) almost all lost contacts at the dimer interface are substituted with equivalent and newly formed interactions inside each monomer, and new stabilizing interactions are formed; and (3) all monomers recreate functional hydrophobic cores. The results of the present study show that both dimeric and monomeric forms of S100 proteins can be functional.     

Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains.

trans activation of promoters by viral regulatory proteins provides a useful tool to study coordinate control of gene expression. Immediate-early (IE) regions 1 and 2 of human cytomegalovirus (CMV) code for a series of proteins that originate from differentially spliced mRNAs. These IE proteins are proposed to regulate the temporal expression of the viral genome. To examine the structure and function of the IE proteins, we used linker insertion mutagenesis of the IE gene region as well as cDNA expression vector cloning of the abundant IE mRNAs. We showed that IE1 and IE2 proteins of CMV exhibit promoter-specific differences in their modes of action by either trans activating early and IE promoters or repressing the major IE promoter (MIEP). Transient cotransfection experiments with permissive human cells revealed a synergistic interaction between the 72- and the 86-kilodalton (kDa) IE proteins in trans activating an early promoter. In addition, transfection studies revealed that the 72-kDa protein was capable of trans activating the MIEP. In contrast, the 86-kDa protein specifically repressed the MIEP and this repression was suppressed by the 72-kDa protein. Furthermore, observations based on the primary sequence structure revealed a modular arrangement of putative regulatory motifs that could either potentiate or repress gene expression. These modular domains are either shared or unique among the IE proteins. From these data, we propose a model for IE protein function in the coordinate control of CMV gene expression.     

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

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 A 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.     

Solution structure of the dimeric SAM domain of MAPKKK Ste11 and its interactions with the adaptor protein Ste50 from the budding yeast: implications for Ste11 activation and signal transmission through the Ste50-Ste11 complex

Ste11, a homologue of mammalian MAPKKKs, together with its binding partner Ste50 works in a number of MAPK signaling pathways of Saccharomyces cerevisiae. Ste11/Ste50 binding is mediated by their sterile alpha motifs or SAM domains, of which homologues are also found in many other intracellular signaling and regulatory proteins. Here, we present the solution structure of the SAM domain or residues D37-R104 of Ste11 and its interactions with the cognate SAM domain-containing region of Ste50, residues M27-Q131. NMR pulse-field-gradient (PFG) and rotational correlation time measurements (tauc) establish that the Ste11 SAM domain exists predominantly as a symmetric dimer in solution. The solution structure of the dimeric Ste11 SAM domain consists of five well-defined helices per monomer packed into a compact globular structure. The dimeric structure of the SAM domain is maintained by a novel dimer interface involving interactions between a number of hydrophobic residues situated on helix 4 and at the beginning of the C-terminal long helix (helix 5). The dimer structure may also be stabilized by potential salt bridge interactions across the interface. NMR H/2H exchange experiments showed that binding of the Ste50 SAM to the Ste11 SAM very likely involves the positively charged extreme C-terminal region as well as exposed hydrophobic patches of the dimeric Ste11 SAM domain. The dimeric structure of the Ste11 SAM and its interactions with the Ste50 SAM may have important roles in the regulation and activation of the Ste11 kinase and signal transmission and amplifications through the Ste50-Ste11 complex.     

Molecular dynamics simulation of dimeric and monomeric forms of human prion protein: insight into dynamics and properties

A central theme in prion protein research is the detection of the process that underlies the conformational transition from the normal cellular prion form (PrP(C)) to its pathogenic isoform (PrP(Sc)). Although the three-dimensional structures of monomeric and dimeric human prion protein (HuPrP) have been revealed by NMR spectroscopy and x-ray crystallography, the process underlying the conformational change from PrP(C) to PrP(Sc) and the dynamics and functions of PrP(C) remain unknown. The dimeric form is thought to play an important role in the conformational transition. In this study, we performed molecular dynamics (MD) simulations on monomeric and dimeric HuPrP at 300 K and 500 K for 10 ns to investigate the differences in the properties of the monomer and the dimer from the perspective of dynamic and structural behaviors. Simulations were also undertaken with Asp178Asn and acidic pH, which is known as a disease-associated factor. Our results indicate that the dynamics of the dimer and monomer were similar (e.g., denaturation of helices and elongation of the beta-sheet). However, additional secondary structure elements formed in the dimer might result in showing the differences in dynamics and properties between the monomer and dimer (e.g., the greater retention of dimeric than monomeric tertiary structure).     

In vivo homodimerisation of HTLV-1 Gag and MA gives clues to the retroviral capsid and TM envelope protein arrangement

During retroviral particle formation, the capsid precursors (Gag) associate with the cell membrane via their matrix (MA) domain to form viral assembling particles. After budding, Gag and its proteolytically matured MA, form a shell in the released immature and mature particles, respectively. Although the arrangement of Gag domains in vitro and their radial organisation in retroviral particles have been extensively studied, little is known concerning Gag inter-subunit interactions in authentic retroviruses. We report that human T-cell leukemia virus type 1 Gag homodimerises in the cell via a disulphide bonding at cysteine 61 in the MA domain. Most Gags are homodimeric after budding and MAs are also dimeric in mature authentic virions. Molecular modelling of the MA domain indicates that non-covalent interactions at the MA dimer interface may also be important for Gag (and MA) dimerisation. In addition, all amino acids previously reported to be involved in MA-transmembrane (TM) interactions are located on the MA face opposite to the dimer interface. The model reveals that homodimerisation is compatible with a hexameric network of Gag and MA dimers that look like the hexameric networks observed for other retroviruses. These data, together with previous studies, lead us to propose a supra-molecular arrangement model in which the transmembrane glycoproteins of the virion envelope are anchored in a hexameric cage hole formed by the MA.     

Conformational stability of dimeric and monomeric forms of the C-terminal domain of human immunodeficiency virus-1 capsid protein

The unfolding equilibrium of the C-terminal domain of human immunodeficiency virus-1 (HIV-1) capsid protein has been analyzed by circular dichroism and fluorescence spectroscopy. The results for the dimeric, natural domain are consistent with a three-state model (N(2)<-->2I<-->2U). The dimer (N(2)) dissociates and partially unfolds in a coupled cooperative process, into a monomeric intermediate (I) of very low conformational stability. This intermediate, which is the only significantly populated form at low (1 microM) protein concentrations, fully preserves the secondary structure but has lost part of the tertiary (intramonomer) interactions found in the dimer. In a second transition, the intermediate cooperatively unfolds into denatured monomer (U). The latter process is the equivalent of a two-state unfolding transition observed for a monomeric domain in which Trp184 at the dimer interface had been truncated to Ala. A highly conserved, disulfide-bonded cysteine, but not the disulfide bond itself, and three conserved residues within the major homology region of the retroviral capsid are important for the conformational stability of the monomer. All these residues are involved also in the association process, despite being located far away from the dimerization interface. It is proposed that dimerization of the C-terminal domain of the HIV-1 capsid protein involves induced-fit recognition, and the conformational reorganization also improves substantially the low intrinsic stability of each monomeric half.