KCTD5 is endowed with large,functionally relevant,interdomain motions

The KCTD family is an emerging class of proteins that are involved in important biological processes whose biochemical and structural properties are rather poorly characterized or even completely undefined. We here used KCTD5, the only member of the family with a known three-dimensional structure, to gain insights into the intrinsic structural stability of the C-terminal domain (CTD) and into the mutual dynamic interplay between the two domains of the protein. Molecular dynamics (MD) simulations indicate that in the simulation timescale (120 ns), the pentameric assembly of the CTD is endowed with a significant intrinsic stability. Moreover, MD analyses also led to the identification of exposed β-strand residues. Being these regions intrinsically sticky, they could be involved in the substrate recognition. More importantly, simulations conducted on the full-length protein provide interesting information of the relative motions between the BTB domain and the CTD of the protein. Indeed, the dissection of the overall motion of the protein is indicative of a large interdomain twisting associated with limited bending movements. Notably, MD data indicate that the entire interdomain motion is pivoted by a single residue (Ser150) of the hinge region that connects the domains. The functional relevance of these motions was evaluated in the context of the functional macromolecular machinery in which KCTD5 is involved. This analysis indicates that the interdomain twisting motion here characterized may be important for the correct positioning of the substrate to be ubiquitinated with respect to the other factors of the ubiquitination machinery.     

MD Simulations of the dsRBP DGCR8 Reveal Correlated Motions that May Aid pri-miRNA Binding

Over the past decade, microRNAs (miRNAs) have been shown to affect gene regulation by basepairing with messenger RNA, and their misregulation has been directly linked with cancer. DGCR8, a protein that contains two dsRNA-binding domains (dsRBDs) in tandem, is vital for nuclear maturation of primary miRNAs (pri-miRNAs) in connection with the RNase III enzyme Drosha. The crystal structure of the DGCR8 Core (493-720) shows a unique, well-ordered structure of the linker region between the two dsRBDs that differs from the flexible linker connecting the two dsRBDs in the antiviral response protein, PKR. To better understand the interfacial interactions between the two dsRBDs, we ran extensive MD simulations of isolated dsRBDs (505-583 and 614-691) and the Core. The simulations reveal correlated reorientations of the two domains relative to one another, with the well-ordered linker and C-terminus serving as a pivot. The results demonstrate that motions at the domain interface dynamically impact the conformation of the RNA-binding surface and may provide an adaptive separation distance that is necessary to allow interactions with a variety of different pri-miRNAs with heterogeneous structures. These results thus provide an entry point for further in vitro studies of the potentially unique RNA-binding mode of DGCR8.     

Sperm coating mechanism from the 1.8 A crystal structure of PDC-109-phosphorylcholine complex

Bovine seminal plasma PDC-109 binds to sperm surface choline lipids and promotes sperm capacitation by stimulating the efflux of cholesterol and phospholipids. The structure of PDC-109 with bound phosphorylcholine was solved using MAD data of a single platinum site. Its two globular (40 x 50 x 20 A(3)) Fn2 domains are linked and clustered by a short polypeptide. The choline binding sites lie at the same face of the molecule. Phosphorylcholine binds to the Fn2 domains through a cation-pi interaction between the quaternary ammonium group and a core tryptophan, plus hydrogen bonding between hydroxyls of exposed tyrosines and the phosphate group. The structure of the PDC-109-oPC complex provides a structural ground for the sperm membrane-coating mechanism underlying PDC-109-induced capacitation.     

Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study

Background

Human blood coagulation factor VIII (fVIII) is a large plasma glycoprotein with sequential domain arrangement in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A1, A2 and A3 domains are interconnected by long linker peptides (a1, a2 and a3) that possess the activation sites. Proteolysis of fVIII zymogen by thrombin or factor Xa results in the generation of the activated form (fVIIIa) which serves as a critical co-factor for factor IXa (fIXa) enzyme in the intrinsic coagulation pathway.

Results

In our efforts to elucidate the structural differences between fVIII and fVIIIa, we developed the solution structural models of both forms, starting from an incomplete 3.7 Å X-ray crystal structure of fVIII zymogen, using explicit solvent MD simulations. The full assembly of B-domainless single-chain fVIII was built between the A1-A2 (Ala1-Arg740) and A3-C1-C2 (Ser1669-Tyr2332) domains. The structural dynamics of fVIII and fVIIIa, simulated for over 70 ns of time scale, enabled us to evaluate the integral motions of the multi-domain assembly of the co-factor and the possible coordination pattern of the functionally important calcium and copper ion binding in the protein.

Conclusions

MD simulations predicted that the acidic linker peptide (a1) between the A1 and A2 domains is largely flexible and appears to mask the exposure of putative fIXa enzyme binding loop (Tyr555-Asp569) region in the A2 domain. The simulation of fVIIIa, generated from the zymogen structure, predicted that the linker peptide (a1) undergoes significant conformational reorganization upon activation by relocating completely to the A1-domain. The conformational transition led to the exposure of the Tyr555-Asp569 loop and the surrounding region in the A2 domain. While the proposed linker peptide conformation is predictive in nature and warrants further experimental validation, the observed conformational differences between the zymogen and activated forms may explain and support the large body of experimental data that implicated the critical importance of the cleavage of the peptide bond between the Arg372 and Ser373 residues for the full co-factor activity of fVIII.     

Conformational dynamics of a multidomain protein by neutron scattering and computational analysis

The flexible conformations of a multidomain protein are responsible for its biological functions. Although MurD, a 47-kDa protein that consists of three domains, sequentially changes its domain conformation from an open form to a closed form through a semiclosed form in its enzymatic reaction, the domain dynamics in each conformation remains unclear. In this study, we verify the conformational dynamics of MurD in the corresponding three states (apo and ATP- and inhibitor-bound states) with a combination of small-angle x-ray and neutron scattering (SAXS and SANS), dynamic light scattering (DLS), neutron backscattering (NBS), neutron spin echo (NSE) spectroscopy, and molecular dynamics (MD) simulations. Applying principal component analysis of the MD trajectories, twisting and open-closed domain modes are identified as the major collective coordinates. The deviations of the experimental SAXS profiles from the theoretical calculations based on the known crystal structures become smaller in the ATP-bound state than in the apo state, and a further decrease is evident upon inhibitor binding. These results suggest that domain motions of the protein are suppressed step by step of each ligand binding. The DLS and NBS data yield collective and self-translational diffusion constants, respectively, and we used them to extract collective domain motions in nanometer and nanosecond scales from the NSE data. In the apo state, MurD shows both twisting and open-closed domain modes, whereas an ATP binding suppresses twisting domain motions, and a further reduction of open-closed mode is seen in the inhibitor-binding state. These observations are consistent with the structure modifications measured by the small-angle scattering as well as the MD simulations. Such changes in the domain dynamics associated with the sequential enzymatic reactions should be related to the affinity and reaction efficiency with a ligand that binds specifically to each reaction state.     

Domain motions of the Mip protein from Legionella pneumophila

The homodimeric 45.6 kDa (total mass) Mip protein, a virulence factor from Legionella pneumophila, was investigated with solution NMR spectroscopy and molecular dynamics (MD) simulations. Two Mip monomers are dimerized via an N-terminal helix bundle that is connected via a long alpha-helix to a C-terminal FKBP domain in each subunit. More than 85% of the amino acids were identified in triple-resonance NMR spectra. (15)N relaxation analysis showed a bimodal distribution of R(1)/R(2) values, with the lower ratio in the N-terminal domain. Relaxation dispersion measurements confirmed that these reduced ratios did not originate from conformational exchange. Thus, two different correlation times (tau(c)) can be deduced, reflecting partly uncoupled motions of both domains. Relaxation data of a Mip(77)(-)(213) monomer mutant were similar to those observed in the dimer, corroborating that the FKBP domain, including part of the connecting helix, behaves as one dynamic entity. MD simulations (18 ns) of the Mip dimer also yielded two different correlation times for the two domains and thus confirm the independence of the domain motions. Principal component analysis of the dihedral space covariance matrix calculated from the MD trajectory suggests a flexible region in the long connecting helix that acts as a hinge between the two domains. Such motion provides a possible explanation of how Mip can bind to complex molecular components of the extracellular matrix and mediate alveolar damage and bacterial spread in the lung.     

A multidomain outer membrane protein from Pasteurella multocida: modelling and simulation studies of PmOmpA

PmOmpA is a two-domain outer membrane protein from Pasteurella multocida. The N-terminal domain of PmOmpA is a homologue of the transmembrane beta-barrel domain of OmpA from Escherichia coli, whilst the C-terminal domain of PmOmpA is a homologue of the extra-membrane Neisseria meningitidis RmpM C-terminal domain. This enables a model of a complete two domain PmOmpA to be constructed and its conformational dynamics explored via MD simulations of the protein embedded within two different phospholipid bilayers (DMPC and DMPE). The conformational stability of the transmembrane beta-barrel is similar to that of a homology model of OprF from Pseudomonas aeruginosa in bilayer simulations. There is a degree of water penetration into the interior of the beta-barrel, suggestive of a possible transmembrane pore. Although the PmOmpA model is stable over 20 ns simulations, retaining its secondary structure and fold integrity throughout, substantial flexibility is observed in a short linker region between the N- and the C-terminal domains. At low ionic strength, the C-terminal domain moves to interact electrostatically with the lipid bilayer headgroups. This study demonstrates that computational approaches may be applied to more complex, multi-domain outer membrane proteins, rather than just to transmembrane beta-barrels, opening the possibility of in silico proteomics approaches to such proteins.     

Inter-domain movements in polyketide synthases: a molecular dynamics study

Insights into the structure and dynamics of modular polyketide synthases (PKS) are essential for understanding the mechanistic details of the biosynthesis of a large number of pharmaceutically important secondary metabolites. The crystal structures of the KS-AT di-domain from erythromycin synthase have revealed the relative orientation of various catalytic domains in a minimal PKS module. However, the relatively large distance between catalytic centers of KS and AT domains in the static structure has posed certain intriguing questions regarding mechanistic details of substrate transfer during polyketide biosynthesis. In order to investigate the role of inter-domain movements in substrate channeling, we have carried out a series of explicit solvent MD simulations for time periods ranging from 10 to 15 ns on the KS-AT di-domain and its sub-fragments. Analyses of these MD trajectories have revealed that both the catalytic domains and the structured inter-domain linker region remain close to their starting structures. Inter-domain movements at KS-linker and linker-AT interfaces occur around hinge regions which connect the structured linker region to the catalytic domains. The KS-linker interface was found to be more flexible compared to the linker-AT interface. However, inter-domain movements observed during the timescale of our simulations do not significantly reduce the distance between catalytic centers of KS and AT domains for facilitating substrate channeling. Based on these studies and prediction of intrinsic disorder we propose that the intrinsically unstructured linker stretch preceding the ACP domain might be facilitating movement of ACP domains to various catalytic centers.     

A multidomain outer membrane protein from Pasteurella multocida: Modelling and simulation studies of PmOmpA

PmOmpA is a two-domain outer membrane protein from Pasteurella multocida. The N-terminal domain of PmOmpA is a homologue of the transmembrane β-barrel domain of OmpA from Escherichia coli, whilst the C-terminal domain of PmOmpA is a homologue of the extra-membrane Neisseria meningitidis RmpM C-terminal domain. This enables a model of a complete two domain PmOmpA to be constructed and its conformational dynamics explored via MD simulations of the protein embedded within two different phospholipid bilayers (DMPC and DMPE). The conformational stability of the transmembrane β-barrel is similar to that of a homology model of OprF from Pseudomonas aeruginosa in bilayer simulations. There is a degree of water penetration into the interior of the β-barrel, suggestive of a possible transmembrane pore. Although the PmOmpA model is stable over 20 ns simulations, retaining its secondary structure and fold integrity throughout, substantial flexibility is observed in a short linker region between the N- and the C-terminal domains. At low ionic strength, the C-terminal domain moves to interact electrostatically with the lipid bilayer headgroups. This study demonstrates that computational approaches may be applied to more complex, multi-domain outer membrane proteins, rather than just to transmembrane β-barrels, opening the possibility of in silico proteomics approaches to such proteins.     

Functional domain motions in proteins on the ~1-100 ns timescale: comparison of neutron spin-echo spectroscopy of phosphoglycerate kinase with molecular-dynamics simulation

Protein function often requires large-scale domain motion. An exciting new development in the experimental characterization of domain motions in proteins is the application of neutron spin-echo spectroscopy (NSE). NSE directly probes coherent (i.e., pair correlated) scattering on the ~1-100 ns timescale. Here, we report on all-atom molecular-dynamics (MD) simulation of a protein, phosphoglycerate kinase, from which we calculate small-angle neutron scattering (SANS) and NSE scattering properties. The simulation-derived and experimental-solution SANS results are in excellent agreement. The contributions of translational and rotational whole-molecule diffusion to the simulation-derived NSE and potential problems in their estimation are examined. Principal component analysis identifies types of domain motion that dominate the internal motion's contribution to the NSE signal, with the largest being classic hinge bending. The associated free-energy profiles are quasiharmonic and the frictional properties correspond to highly overdamped motion. The amplitudes of the motions derived by MD are smaller than those derived from the experimental analysis, and possible reasons for this difference are discussed. The MD results confirm that a significant component of the NSE arises from internal dynamics. They also demonstrate that the combination of NSE with MD is potentially useful for determining the forms, potentials of mean force, and time dependence of functional domain motions in proteins.     

Bending of the calmodulin central helix: a theoretical study.

The crystal structure of calcium-calmodulin (CaM) reveals a protein with a typical dumbbell structure. Various spectroscopic studies have suggested that the central linker region of CaM, which is alpha-helical in the crystal structure, is flexible in solution. In particular, NMR studies have indicated the presence of a flexible backbone between residues Lys 77 and Asp 80. This flexibility is related directly to the function of the protein because it enables the N- and C-terminal domains of the protein to move toward each other and bind to the CaM-binding domain of a target protein. We have investigated the flexibility of the CaM central helix by a variety of computational techniques: molecular dynamics (MD) simulations, normal mode analysis (NMA), and essential dynamics (ED) analysis. Our MD results reproduce the experimentally determined location of the bend in a simulation of only the CaM central helix, indicating that the bending point is an intrinsic property of the alpha-helix, for which the remainder of the protein is not important. Interestingly, the modes found by the ED analysis of the MD trajectory are very similar to the lowest frequency modes from the NM analysis and to modes found by an ED analysis of different structures in a set of NMR structures. Electrostatic interactions involving residues Arg 74 and Asp 80 seem to be important for these bending motions and unfolding, which is in line with pH-dependent NMR and CD studies.     

Effects of Pro1266Leu mutation on structure and function of glycoprotein Ib binding domain of von Willebrand factor

Glycoprotein Ibα (GpIbα) binding ability of A1 domain of von Willebrand factor (vWF) facilitates platelet adhesion that plays a crucial role in maintaining hemostasis and thrombosis at the site of vascular damage. There are both “loss as well as gain of function” mutations observed in this domain. Naturally occurring “gain of function” mutations leave self-activating impacts on the A1 domain which turns the normal binding to characteristic constitutive binding with GPIbα. These “gain of function” mutations are associated with the von Willebrand disease type 2B. In recent years, studies focused on understanding the mechanism and conformational patterns attached to these phenomena have been conducted, but the conformational pathways leading to such binding patterns are poorly understood as of now. To obtain a microscopic picture of such events for the better understanding of pathways, we used molecular dynamics (MD) simulations along with principal component analysis and normal mode analysis to study the effects of Pro1266Leu (Pro503Leu in structural context) mutation on the structure and function of A1 domain of vWF. MD simulations have provided atomic-level details of intermolecular motions as a function of time to understand the dynamic behavior of A1 domain of vWF. Comparative analysis of the trajectories obtained from MD simulations of both the wild type and Pro503Leu mutant suggesting appreciable conformational changes in the structure of mutant which might provide a basis for assuming the “gain of function” effects of these mutations on the A1 domain of vWF, resulting in the constitutive binding with GpIbα.     

Armadillo: domain boundary prediction by amino acid composition

The identification and annotation of protein domains provides a critical step in the accurate determination of molecular function. Both computational and experimental methods of protein structure determination may be deterred by large multi-domain proteins or flexible linker regions. Knowledge of domains and their boundaries may reduce the experimental cost of protein structure determination by allowing researchers to work on a set of smaller and possibly more successful alternatives. Current domain prediction methods often rely on sequence similarity to conserved domains and as such are poorly suited to detect domain structure in poorly conserved or orphan proteins. We present here a simple computational method to identify protein domain linkers and their boundaries from sequence information alone. Our domain predictor, Armadillo (http://armadillo.blueprint.org), uses any amino acid index to convert a protein sequence to a smoothed numeric profile from which domains and domain boundaries may be predicted. We derived an amino acid index called the domain linker propensity index (DLI) from the amino acid composition of domain linkers using a non-redundant structure dataset. The index indicates that Pro and Gly show a propensity for linker residues while small hydrophobic residues do not. Armadillo predicts domain linker boundaries from Z-score distributions and obtains 35% sensitivity with DLI in a two-domain, single-linker dataset (within +/-20 residues from linker). The combination of DLI and an entropy-based amino acid index increases the overall Armadillo sensitivity to 56% for two domain proteins. Moreover, Armadillo achieves 37% sensitivity for multi-domain proteins, surpassing most other prediction methods. Armadillo provides a simple, but effective method by which prediction of domain boundaries can be obtained with reasonable sensitivity. Armadillo should prove to be a valuable tool for rapidly delineating protein domains in poorly conserved proteins or those with no sequence neighbors. As a first-line predictor, domain meta-predictors could yield improved results with Armadillo predictions.     

Characteristics and prediction of domain linker sequences in multi-domain proteins

To facilitate swift structural characterizations, structural genomic/proteomic projects need to divide large multi-domain proteins into structural domains and to determine their structures separately. Thus, the assignment of structural domains based solely on sequence information, especially on the physico-chemical properties of the amino acid sequences, could be very helpful for such projects. In this study, we examined the characteristics of domain linker sequences, which are loop sequences connecting two structural domains. To this end, we prepared a set of 101 non-redundant multi-domain protein sequences with known structures, and performed an analysis of the linker sequences. The analysis revealed that the frequencies of five (Pro, Gly, Asp, Asn, Lys) amino acid residues differed significantly between the linker and non-linker loop sequences. Moreover, we observed a similar deviation for the residue pair frequencies between the two types of loop sequences. Finally, we describe an automated method, based on the above analysis, to detect loops that have high probabilities of being domain linkers in a protein sequence.     

The influence of interdomain interactions on the intradomain motions in yeast phosphoglycerate kinase: a molecular dynamics study

A 3-ns molecular dynamics simulation in explicit solvent was performed to examine the inter- and intradomain motions of the two-domain enzyme yeast phosphoglycerate kinase without the presence of substrates. To elucidate contributions from individual domains, simulations were carried out on the complete enzyme as well as on each isolated domain. The enzyme is known to undergo a hinge-bending type of motion as it cycles from an open to a closed conformation to allow the phosphoryl transfer occur. Analysis of the correlation of atomic movements during the simulations confirms hinge bending in the nanosecond timescale: the two domains of the complete enzyme exhibit rigid body motions anticorrelated with respect to each other. The correlation of the intradomain motions of both domains converges, yielding a distinct correlation map in the enzyme. In the isolated domain simulations—in which interdomain interactions cannot occur—the correlation of domain motions no longer converges and shows a very small correlation during the same simulation time. This result points to the importance of interdomain contacts in the overall dynamics of the protein. The secondary structure elements responsible for interdomain contacts are also discussed.     

Folding and Misfolding of a PDZ Tandem Repeat

Although the vast majority of the human proteome is represented by multi-domain proteins, the study of multi-domain folding and misfolding is a relatively poorly explored field. The protein Whirlin is a multi-domain scaffolding protein expressed in the inner ear. It is characterized by the presence of tandem repeats of PDZ domains. The first two PDZ domains of Whirlin (PDZ1 and PDZ2 – namely P1P2) are structurally close and separated by a disordered short linker. We recently described the folding mechanism of the P1P2 tandem. The difference in thermodynamic stability of the two domains allowed us to selectively unfold one or both PDZ domains and to pinpoint the accumulation of a misfolded intermediate, which we demonstrated to retain physiological binding activity. In this work, we provide an extensive characterization of the folding and unfolding of P1P2. Based on the observed data, we describe an integrated kinetic analysis that satisfactorily fits the experiments and provides a valuable model to interpret multi-domain folding. The experimental and analytical approaches described in this study may be of general interest for the interpretation of complex multi-domain protein folding kinetics.     

Molecular dynamics study of conformational changes in human serum albumin by binding of fatty acids

Human serum albumin (HSA) binds with fatty acids under normal physiologic conditions. To date, there is little published information on the tertiary structure of HSA-fatty acid complex in aqueous solution. In the present study, we used molecular dynamics (MD) simulations to elucidate possible structural changes of HSA brought about by the binding of fatty acids. Both unliganded HSA and HSA-fatty acid complex models for MD calculations were constructed based on the X-ray crystal structures. Five myristates (MYRs) were bound in the HSA-fatty acid complex model. In the present MD study, the motion of domains I and III caused by the binding of MYR molecules increased the radius of gyration of HSA. Root-mean-square fluctuations from the MD simulations revealed that the atomic fluctuations of the specific amino acids at drug-binding site I that can regulate the drug-binding affinity were increased by the binding of MYR molecules. Primary internal motions, characterized by the first three principal components, were observed mainly at domains I and III in the principal component analysis for trajectory data. The directional motion projected on the first principal component of unliganded HSA was conserved in HSA-MYR complex as the third principal directional motion with higher frequency. However, the third principal directional motion in unliganded HSA turned into the first principal directional motion with lower frequency in the HSA-MYR complex. Thus, the present MD study provides insights into the possible conformational changes of HSA caused by the binding of fatty acids.