Network model of a protein globule

Phase transition of a protein globule is considered in the frameworks of (i) the generalized mean-field theory for the order parameter, characterizing the extent of the deviation of a protein three-dimensional structure from its native state and (ii) the network model that treats a protein globule as a small-world network with a significant percent of long-range links between amino acid residues. Temperature dependencies of the introduced order parameter are defined and phase-transition temperatures are found on the basis of the function defining the distribution of links’ numbers for protein residues. An important role of long-range links, promoting considerable rise of thermal protein stability, is demonstrated by the example of a correlation between protein melting temperature and a fraction of disulfide bonds.     

Integrating molecular dynamics and co-evolutionary analysis for reliable target prediction and deregulation of the allosteric inhibition of aspartokinase for amino acid production

Deregulation of allosteric inhibition of enzymes is a challenge for strain engineering and has been achieved so far primarily by random mutation and trial-and-error. In this work, we used aspartokinase, an important allosteric enzyme for industrial amino acids production, to demonstrate a predictive approach that combines protein dynamics and evolution for a rational reengineering of enzyme allostery. Molecular dynamic simulation of aspartokinase III (AK3) from Escherichia coli and statistical coupling analysis of protein sequences of the aspartokinase family allowed to identify a cluster of residues which are correlated during protein motion and coupled during the evolution. This cluster of residues forms an interconnected network mediating the allosteric regulation, including most of the previously reported positions mutated in feedback insensitive AK3 mutants. Beyond these mutation positions, we have successfully constructed another twelve targeted mutations of AK3 desensitized toward lysine inhibition. Six threonine-insensitive mutants of aspartokinase I-homoserine dehydrogenase I (AK1-HD1) were also created based on the predictions. The proposed approach can be widely applied for the deregulation of other allosteric enzymes.     

Integrating protein structural dynamics and evolutionary analysis with Bio3D

Background

Popular bioinformatics approaches for studying protein functional dynamics include comparisons of crystallographic structures, molecular dynamics simulations and normal mode analysis. However, determining how observed displacements and predicted motions from these traditionally separate analyses relate to each other, as well as to the evolution of sequence, structure and function within large protein families, remains a considerable challenge. This is in part due to the general lack of tools that integrate information of molecular structure, dynamics and evolution.

Results

Here, we describe the integration of new methodologies for evolutionary sequence, structure and simulation analysis into the Bio3D package. This major update includes unique high-throughput normal mode analysis for examining and contrasting the dynamics of related proteins with non-identical sequences and structures, as well as new methods for quantifying dynamical couplings and their residue-wise dissection from correlation network analysis. These new methodologies are integrated with major biomolecular databases as well as established methods for evolutionary sequence and comparative structural analysis. New functionality for directly comparing results derived from normal modes, molecular dynamics and principal component analysis of heterogeneous experimental structure distributions is also included. We demonstrate these integrated capabilities with example applications to dihydrofolate reductase and heterotrimeric G-protein families along with a discussion of the mechanistic insight provided in each case.

Conclusions

The integration of structural dynamics and evolutionary analysis in Bio3D enables researchers to go beyond a prediction of single protein dynamics to investigate dynamical features across large protein families. The Bio3D package is distributed with full source code and extensive documentation as a platform independent R package under a GPL2 license from http://thegrantlab.org/bio3d/.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0399-6) contains supplementary material, which is available to authorized users.     

Understanding the role of hydrogen bonds in water dynamics and protein stability

The mechanisms of cold and pressure denaturation of proteins are a matter of debate, but it is commonly accepted that water plays a fundamental role in the process. It has been proposed that the denaturation process is related to an increase of hydrogen bonds among hydration water molecules. Other theories suggest that the causes of denaturation are the density fluctuations of surface water, or the destabilization of hydrophobic contacts as a consequence of water molecule inclusions inside the protein, especially at high pressures. We review some theories that have been proposed to give insight into this problem, and we describe a coarse-grained model of water that compares well with experiments for proteins’ hydration water. We introduce its extension for a homopolymer in contact with the water monolayer and study it by Monte Carlo simulations in an attempt to understand how the interplay of water cooperativity and interfacial hydrogen bonds affects protein stability.     

Oligomers of the Cdc7/Dbf4 protein kinase exist in the yeast cell

Cdc7/Dbf4 protein kinase is required for the initiation of DNA replication in Saccharomyces cerevisiae. Cdc7/Dbf4 protein kinase is not a cyclin-dependent kinase (CDK), but is regulated in a similar fashion in that the Cdc7 kinase subunit is inactive in the absence of the regulatory subunit Dbf4. In contrast to what is known about CDKs, Cdc7/Dbf4 protein kinase is shown to be an oligomer in the cell in this report. Genetic data that support this claim include interallelic complementation between several cdc7ts alleles and the cdc7T281A allele and also the results of experiments using the two-hybrid system with Cdc7 in both DNA-binding and transactivation domain plasmids. A molecular interaction between two different Cdc7 molecules was shown by using a HA-tagged Cdc7 protein that differs in size from the wild-type Cdc7 protein: an anti-HA antibody immunoprecipitates both proteins in appproximately equal stoichiometry. Analysis of the native molecular weight of Cdc7/Dbf4 protein kinase is consistent with oligomerization of the Cdc7 protein in that complexes of about 180 and 300 kDa were found. Oligomers of Cdc7 protein may exist for the purpose of allosteric regulation or to allow phosphorylation of multiple substrate protein molecules. Received: 4 January 1998 / Accepted: 16 June 1998     

Evolution of domain combinations in protein kinases and its implications for functional diversity

Protein kinases phosphorylating Ser/Thr/Tyr residues in several cellular proteins exert tight control over their biological functions. They constitute the largest protein family in most eukaryotic species. Protein kinases classified based on sequence similarity in their catalytic domains, cluster into subfamilies, which share gross functional properties. Many protein kinases are associated or tethered covalently to domains that serve as adapter or regulatory modules, aiding substrate recruitment, specificity, and also serve as scaffolds. Hence the modular organisation of the protein kinases serves as guidelines to their functional and molecular properties. Analysis of genomic repertoires of protein kinases in eukaryotes have revealed wide spectrum of domain organisation across various subfamilies of kinases. Occurrence of organism-specific novel domain combinations suggests functional diversity achieved by protein kinases in order to regulate variety of biological processes. In addition, domain architecture of protein kinases revealed existence of hybrid protein kinase subfamilies and their emerging roles in the signaling of eukaryotic organisms. In this review we discuss the repertoire of non-kinase domains tethered to multi-domain kinases in the metazoans. Similarities and differences in the domain architectures of protein kinases in these organisms indicate conserved and unique features that are critical to functional specialization.     

Mechanism for CARMIL protein inhibition of heterodimeric actin-capping protein

Capping protein (CP) controls the polymerization of actin filaments by capping their barbed ends. In lamellipodia, CP dissociates from the actin cytoskeleton rapidly, suggesting the possible existence of an uncapping factor, for which the protein CARMIL (capping protein, Arp2/3 and myosin-I linker) is a candidate. CARMIL binds to CP via two motifs. One, the CP interaction (CPI) motif, is found in a number of unrelated proteins; the other motif is unique to CARMILs, the CARMIL-specific interaction motif. A 115-aa CARMIL fragment of CARMIL with both motifs, termed the CP-binding region (CBR), binds to CP with high affinity, inhibits capping, and causes uncapping. We wanted to understand the structural basis for this function. We used a collection of mutants affecting the actin-binding surface of CP to test the possibility of a steric-blocking model, which remained open because a region of CBR was not resolved in the CBR/CP co-crystal structure. The CP actin-binding mutants bound CBR normally. In addition, a CBR mutant with all residues of the unresolved region changed showed nearly normal binding to CP. Having ruled out a steric blocking model, we tested an allosteric model with molecular dynamics. We found that CBR binding induces changes in the conformation of the actin-binding surface of CP. In addition, ～30-aa truncations on the actin-binding surface of CP decreased the affinity of CBR for CP. Thus, CARMIL promotes uncapping by binding to a freely accessible site on CP bound to a filament barbed end and inducing a change in the conformation of the actin-binding surface of CP.     

Disruption of quaternary structure in Escherichia coli dihydrodipicolinate synthase (DHDPS) generates a functional monomer that is no longer inhibited by lysine

Escherichia coli dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52), a natively homotetrameric enzyme was converted to a monomeric species through the introduction of destabilising interactions at two different subunit interfaces allowing exploration of the roles of the quaternary structure in affecting catalytic competency. The double mutant DHDPS-L197D/Y107W displays gel filtration characteristics consistent with a single non-interacting monomeric species, which was confirmed by sedimentary velocity experiments. This monomer was shown to be catalytically active, but with reduced catalytic efficiency (k_cat = 9.8 ± 0.5 s⁻¹), displaying 8% of the specific activity of the wild-type enzyme. The Michaelis constants for the substrates pyruvate and for (S)-aspartate semialdehyde increased by an order of magnitude, indicating that quaternary structure plays a significant role in substrate specificity. This monomeric species exhibited an enhanced propensity for aggregation and inactivation, indicating that whilst the oligomerization is not an intrinsic criterion for catalysis, higher oligomeric forms may benefit from both increased catalytic efficiency and diminished aggregation propensity. Furthermore, allosteric inhibition by (S)-lysine was abolished for DHDPS-L197D/Y107W, confirming the importance of the dimeric unit as the minimal functional assembly for efficient (S)-lysine binding.     

Rat Brain Protein Kinase C: Purification, Antibody Production, and Quantification in Discrete Regions of Hippocampus

Protein kinase C (PKC), a calcium- and phospholipid-dependent kinase, is highly enriched in rat brain, where it may function in signal transduction processes. We purified rat brain PKC to homogeneity by a three-column procedure of diethylaminoethyl-cellulose, phenyl-Sepharose, and protamine-agarose with a yield of 16% and a final specific activity of 9,600 pmol of [3H]phorbol-12,13-dibutyrate bound/mg of protein. The pure protein consisted of a doublet of 80 and 78 kilodaltons. Rabbit antibodies prepared against a beta-type PKC synthetic peptide sequence (RAKIGQGTKAPEEKTANTISK) showed high specificity and sensitivity for PKC and recognized only the 78-kilodalton form of PKC. Micropunches (300 microns in diameter) of rat hippocampal subregions were solubilized in sodium dodecyl sulfate (SDS) sample buffer, electrophoresed on SDS-10% polyacrylamide gels, and transferred to nitrocellulose. PKC was visualized by 125I-protein A autoradiography and quantified by densitometry. The highest concentrations of PKC were found in the CA1 pyramidal cell layer (0.43 +/- 0.04 OD), with the lowest amounts in the CA3 and CA4 pyramidal cell layers (0.11 +/- 0.02 and 0.085 +/- 0.006 OD, respectively). These results demonstrate a simple way of preparing antibodies against domains of PKC. We also describe a procedure for quantifying the relative amounts of PKC in discrete brain regions.     

Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers

Chimeric Ca²⁺/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca²⁺ and Ca²⁺/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca²⁺-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the β-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by β-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca²⁺ concentration in target cells, thereby controlling developmental events in the anther. Received: 29 January 1999 / Accepted: 12 February 1999     

HIV-1 TAT-mediated protein transduction of human HPRT into deficient cells

Lesch–Nyhan disease (LND) is a severe and incurable X-linked genetic syndrome caused by the deficiency of hypoxanthine–guanine phosphoribosyltransferase (HPRT), resulting in severe alterations of central nervous system, hyperuricemia and subsequent impaired renal functions. Therapeutic options consist in supportive care and treatments of complications, but the disease remains largely untreatable. Enzyme replacement of the malfunctioning cytosolic protein might represent a possible therapeutic approach for the LND treatment. Protein transduction domains, such as the TAT peptide derived from HIV TAT protein, have been used to transduce macromolecules into cells in vitro and in vivo. The present study was aimed to the generation of TAT peptide fused to human HPRT for cell transduction in enzyme deficient cells. Here we document the construction, expression and delivery of a functional HPRT enzyme into deficient cells by TAT transduction domain and by liposome mediated protein transfer. With this approach we demonstrate the correction of the enzymatic defect in HPRT deficient cells.     

Integration and channeling of calcium signaling through the CBL calcium sensor/CIPK protein kinase network

Plant development and reproduction depend on a precise recognition of environmental conditions and the integration of this information with endogenous metabolic and developmental cues. Calcium ions have been firmly established as ubiquitous second messengers functioning in these processes. Calcium signal deciphering and signal-response coupling often involve calcium-binding proteins as responders or relays in this information flow. Here we review the calcineurin B-like protein (CBL) calcium sensor/CBL-interacting protein kinase (CIPK) network as a newly emerging signaling system mediating a complex array of environmental stimuli. We focus particularly on the mechanisms generating signaling specificity. Moreover, we emphasize the functional implications that are emerging from the analyses of CBL and CIPK loss-of-function mutants.     

The crystal complex of phosphofructokinase-2 of Escherichia coli with fructose-6-phosphate: kinetic and structural analysis of the allosteric ATP inhibition

Substrate inhibition by ATP is a regulatory feature of the phosphofructokinases isoenzymes from Escherichia coli (Pfk-1 and Pfk-2). Under gluconeogenic conditions, the loss of this regulation in Pfk-2 causes substrate cycling of fructose-6-phosphate (fructose-6-P) and futile consumption of ATP delaying growth. In the present work, we have broached the mechanism of ATP-induced inhibition of Pfk-2 from both structural and kinetic perspectives. The crystal structure of Pfk-2 in complex with fructose-6-P is reported to a resolution of 2 Å. The comparison of this structure with the previously reported inhibited form of the enzyme suggests a negative interplay between fructose-6-P binding and allosteric binding of MgATP. Initial velocity experiments show a linear increase of the apparent K_0.5 for fructose-6-P and a decrease in the apparent k_cat as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (n_H of approximately 2). Differences and resemblances in the patterns of fructose-6-P binding and the mechanism of inhibition are discussed for Pfk-1 and Pfk-2, as an example of evolutionary convergence, because these enzymes do not share a common ancestor.     

MAPK and PI3K activities are required for leptin stimulation of protein synthesis in human trophoblastic cells

Leptin, the LEP gene product, is produced in placenta where it has been found to be an important autocrine signal for trophoblastic growth during pregnancy. Thus, we have recently described the antiapoptotic and trophic effect of leptin on choriocarcinoma cell line JEG-3, stimulating DNA and protein synthesis. We have also demonstrated the presence of leptin receptor and leptin signaling in normal human trophoblastic cells, activating JAK-STAT, PI3K and MAPK pathways. In the present work we have employed dominant negative forms of MAPK and PKB constructs to find out the signaling pathways that specifically mediates the effect of leptin on protein synthesis. As previously shown, leptin stimulates protein synthesis as assessed by ³H-leucine incorporation. However, both dominant negative forms of MAPK and PKB inhibited protein synthesis in JEG-3 choriocarcinoma cells. The inhibition of PKB and MAPK activity by transfection with the dominant negative kinases prevented the leptin stimulation of p70 S6K, which is known to be an important kinase in the regulation of protein synthesis. Moreover, leptin stimulation of phosphorylation of EIF4EBP1 and EIF4E, which allows the initiation of translation was also prevented by MAPK and PI3K dominant negative constructs. Therefore, these results demonstrate that both PI3K and MAPK are necessary to observe the effect of leptin signaling that mediates protein synthesis in choriocarcinoma cells JEG-3.     

The pH-dependent unfolding mechanism of P2 myelin protein: an experimental and computational study

The P2 protein is a small, extrinsic protein of the myelin membrane in the peripheral nervous system that structurally belongs to the fatty acid binding proteins (FABPs) family, sharing with them a 10 strands beta-barrel structure. FABPs appear to be involved in cellular fatty acid transport, but very little is known about the role of P2 in the metabolism of peripheral myelin lipids. Study of protein conformation at different pHs is a useful tool for the characterization of the unfolding mechanisms and the intrinsic conformational properties of the protein, and may give insight into factors that guide protein folding pathways. In particular, low pH conditions have been shown to induce partially folded states in several proteins. In this paper, the acidic unfolding of purified P2 protein was studied with both spectroscopic techniques and molecular dynamics simulation. Both experimental and computational results indicate the presence of a partly folded state at low pH, which shows structural changes mainly involving the lid that is formed by the helix-turn-helix domain. The opening of the lid, together with a barrel relaxation, could regulate the ligand exchanges near the cell membrane, supporting the hypothesis that the P2 protein may transport fatty acids between Schwann cells and peripheral myelin.     

The use of ionisation constants of amino acids for protein signal analysis within the resonant recognition model--application to proteases

Biological functions of proteins and their active 3D structures are determined by the linear sequences of amino acids. The resonant recognition model (RRM) is a physico-mathematical model developed for structure/function analysis of protein and DNA sequences. Here, we are comparing results of the RRM analysis [1,2] of protease proteins using the electron-ion interaction potential (EIIP) and ionisation constant (IC) of amino acids. The results obtained reveal that the IC parameter can be successfully used to determine the characteristic patterns of different functional protease subgroups.     

Improved intracellular delivery of glucocerebrosidase mediated by the HIV-1 TAT protein transduction domain

Enzyme replacement therapy (ERT) for Gaucher disease designed to target glucocerebrosidase (GC) to macrophages via mannose-specific endocytosis is very effective in reversing hepatosplenomegaly, and normalizing hematologic parameters but is less effective in improving bone and lung involvement and ineffective in brain. Recombinant GCs containing an in-frame fusion to the HIV-1 trans-activator protein transduction domain (TAT) were expressed in eukaryotic cells in order to obtain active, normally glycosylated GC fusion proteins for enzyme uptake studies. Despite the absence of mannose-specific endocytic receptors on the plasma membranes of various fibroblasts, the recombinant GCs with C-terminal TAT fusions were readily internalized by these cells. Immunofluorescent confocal microscopy demonstrated the recombinant TAT-fusion proteins with a mixed endosomal and lysosomal localization. Thus, TAT-modified GCs represent a novel strategy for a new generation of therapeutic enzymes for ERT for Gaucher disease.