Structured and Unstructured Binding of an Intrinsically Disordered Protein as Revealed by Atomistic Simulations.

Intrinsically disordered proteins (IDPs) are a set of proteins that lack a definite secondary structure in solution. IDPs can acquire tertiary structure when bound to their partners; therefore, the recognition process must also involve protein folding. The nature of the transition state (TS), structured or unstructured, determines the binding mechanism. The characterization of the TS has become a major challenge for experimental techniques and molecular simulations approaches since diffusion, recognition, and binding is coupled to folding. In this work we present atomistic molecular dynamics (MD) simulations that sample the free energy surface of the coupled folding and binding of the transcription factor c-myb to the cotranscription factor CREB binding protein (CBP). This process has been recently studied and became a model to study IDPs. Despite the plethora of available information, we still do not know how c-myb binds to CBP. We performed a set of atomistic biased MD simulations running a total of 15.6 µs. Our results show that c-myb folds very fast upon binding to CBP with no unique pathway for binding. The process can proceed through both structured or unstructured TS's with similar probabilities. This finding reconciles previous seemingly different experimental results. We also performed Go-type coarse-grained MD of several structured and unstructured models that indicate that coupled folding and binding follows a native contact mechanism. To the best of our knowledge, this is the first atomistic MD simulation that samples the free energy surface of the coupled folding and binding processes of IDPs.

Proteome scale comparative modeling for conserved drug and vaccine targets identification in Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis (Cp) is a pathogenic bacterium that causes caseous lymphadenitis (CLA), ulcerative lymphangitis, mastitis, and edematous to a broad spectrum of hosts, including ruminants, thereby threatening economic and dairy industries worldwide. Currently there is no effective drug or vaccine available against Cp. To identify new targets, we adopted a novel integrative strategy, which began with the prediction of the modelome (tridimensional protein structures for the proteome of an organism, generated through comparative modeling) for 15 previously sequenced C. pseudotuberculosis strains. This pan-modelomics approach identified a set of 331 conserved proteins having 95-100% intra-species sequence similarity. Next, we combined subtractive proteomics and modelomics to reveal a set of 10 Cp proteins, which may be essential for the bacteria. Of these, 4 proteins (tcsR, mtrA, nrdI, and ispH) were essential and non-host homologs (considering man, horse, cow and sheep as hosts) and satisfied all criteria of being putative targets. Additionally, we subjected these 4 proteins to virtual screening of a drug-like compound library. In all cases, molecules predicted to form favorable interactions and which showed high complementarity to the target were found among the top ranking compounds. The remaining 6 essential proteins (adk, gapA, glyA, fumC, gnd, and aspA) have homologs in the host proteomes. Their active site cavities were compared to the respective cavities in host proteins. We propose that some of these proteins can be selectively targeted using structure-based drug design approaches (SBDD). Our results facilitate the selection of C. pseudotuberculosis putative proteins for developing broad-spectrum novel drugs and vaccines. A few of the targets identified here have been validated in other microorganisms, suggesting that our modelome strategy is effective and can also be applicable to other pathogens.

How mitogen-activated protein kinases recognize and phosphorylate their targets: A QM/MM study.

Mitogen-activated protein kinase (MAPK) signaling pathways play an essential role in the transduction of environmental stimuli to the nucleus, thereby regulating a variety of cellular processes, including cell proliferation, differentiation, and programmed cell death. The components of the MAPK extracellular activated protein kinase (ERK) cascade represent attractive targets for cancer therapy, as their aberrant activation is a frequent event among highly prevalent human cancers. To understand how MAPKs recognize and phosphorylate their targets is key to unravel their function. However, these events are still poorly understood because of the lack of complex structures of MAPKs with their bound targets in the active site. Here we have modeled the interaction of ERK with a target peptide and analyzed the specificity toward Ser/Thr-Pro motifs. By using a quantum mechanics/molecular mechanics (QM/MM) approach, we propose a mechanism for the phosphoryl transfer catalyzed by ERK that offers new insights into MAPK function. Our results suggest that (1) the proline residue has a role in both specificity and phospho transfer efficiency, (2) the reaction occurs in one step, with ERK2 Asp(147) acting as the catalytic base, (3) a conserved Lys in the kinase superfamily that is usually mutated to check kinase activity strongly stabilizes the transition state, and (4) the reaction mechanism is similar with either one or two Mg(2+) ions in the active site. Taken together, our results provide a detailed description of the molecular events involved in the phosphorylation reaction catalyzed by MAPK and contribute to the general understanding of kinase activity.

Binding-induced folding of a natively unstructured transcription factor.

Transcription factors are central components of the intracellular regulatory networks that control gene expression. An increasingly recognized phenomenon among human transcription factors is the formation of structure upon target binding. Here, we study the folding and binding of the pKID domain of CREB to the KIX domain of the co-activator CBP. Our simulations of a topology-based Go-type model predict a coupled folding and binding mechanism, and the existence of partially bound intermediates. From transition-path and Phi-value analyses, we find that the binding transition state resembles the unstructured state in solution, implying that CREB becomes structured only after committing to binding. A change of structure following binding is reminiscent of an induced-fit mechanism and contrasts with models in which binding occurs to pre-structured conformations that exist in the unbound state at equilibrium. Interestingly, increasing the amount of structure in the unbound pKID reduces the rate of binding, suggesting a "fly-casting"-like process. We find that the inclusion of attractive non-native interactions results in the formation of non-specific encounter complexes that enhance the on-rate of binding, but do not significantly change the binding mechanism. Our study helps explain how being unstructured can confer an advantage in protein target recognition. The simulations are in general agreement with the results of a recently reported nuclear magnetic resonance study, and aid in the interpretation of the experimental binding kinetics.

Reverse Engineering of an Aspirin-Responsive Transcriptional Regulator in Escherichia coli.

Bacterial transcription factors (TFs) are key devices for the engineering of complex circuits in many biotechnological applications, yet there are few well-characterized inducer-responsive TFs that could be used in the context of an animal or human host. We have deciphered the inducer recognition mechanism of two AraC/XylS regulators from Pseudomonas putida (BenR and XylS) for creating a novel expression system responsive to acetyl salicylate (i.e., aspirin). Using protein homology modeling and molecular docking with the cognate inducer benzoate and a suite of chemical analogues, we identified the conserved binding pocket of BenR and XylS. By means of site-directed mutagenesis, we identified a single amino acid position required for efficient inducer recognition and transcriptional activation. Whereas this modification in BenR abolishes protein activity, in XylS, it increases the response to several inducers, including acetyl salicylic acid, to levels close to those achieved by the canonical inducer. Moreover, by constructing chimeric proteins with swapped N-terminal domains, we created novel regulators with mixed promoter and inducer recognition profiles. As a result, a collection of engineered TFs was generated with an enhanced response to benzoate, 3-methylbenzoate, 2-methylbenzoate, 4-methylbenzoate, salicylic acid, aspirin, and acetylsalicylic acid molecules for eliciting gene expression in E. coli.

Investigation of the catalytic mechanism of farnesyl pyrophosphate synthase by computer simulation.

Farnesyl pyrophosphate synthase (FPPS) catalyses the formation of a key cellular intermediate in isoprenoid metabolic pathways, farnesyl pyrophosphate, by the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP). Recently, FPPS has been shown to represent an important target for the treatment of parasitic diseases such as Chagas disease and African trypanosomiasis. Bisphosphonates, pyrophosphate analogues in which the oxygen bridge between the two phosphorus atoms has been replaced by a carbon substituted with different side chains, are able to inhibit the FPPS enzyme. Moreover, nitrogen-containing bisphosphonates have been proposed as carbocation transition state analogues of FPPS. On the basis of structural and kinetic data, different catalytic mechanisms have been proposed for FPPS. By analyzing different reaction coordinates we propose that the reaction occurs in one step through a carbocationic transition state and the subsequent transfer of a hydrogen atom from IPP to the pyrophosphate moiety of DMAPP. Moreover, we have analyzed the role of the active site amino acids on the activation barrier and the reaction mechanism. The structure of the active site is well conserved in the isoprenyl diphosphate synthase family; thus, our results are relevant for the understanding of this important class of enzymes and for the design of more potent and specific inhibitors for the treatment of parasitic diseases.

Characterization of the farnesyl pyrophosphate synthase of Trypanosoma cruzi by homology modeling and molecular dynamics.

Chagas' disease, caused by the Trypanosoma cruzi parasite, is one of the largest public health problems in the Western hemisphere, with 16-18 million people infected, and approximately 100 million people at risk. Many efforts towards the development of targeted antiparasitic agents have recently been described. Of interest, bisphosphonates, pyrophosphate analogs in which the oxygen bridge between the two phosphorus atoms has been replaced by a carbon substituted with different side chains, are able to inhibit the growth of T. cruzi. The enzyme T. cruzi farnesyl pyrophosphate synthase (TcFPPS) involved in the mevalonate pathway, has been recently identified as the target of bisphosphonates. The protein has 362 amino acids and a molecular mass of 41.2 kDa. Several sequence motifs found in other FPPSs are present in TcFPPS. In this study we have modeled the structure of TcFPPS based on the structure of the avian FPPS. We have characterized the interaction with its substrates, isopentyl pyrophosphate and dimethylallyl pyrophosphate, and the mechanism of inhibition by the potent bisphosphonate risedronate (K(i) of 0.032+/-0.002 microM) by means of molecular dynamics techniques. We propose that homorisedronate, which has an extra methylene and a K(i) of 8.17+/-1.36 microM, does not form strong hydrogen bonds with TYR 211 and THR 208, which may be responsible for its lower activity as compared to risedronate. Moreover, we were able to reproduce the structural changes that occur upon the binding of the third Mg2+ to the active site of the protein. Taken together, our results provide a structural model for the design of novel inhibitors that may prove useful for the treatment of Chagas' disease.

Phosphorylation Regulates the Bound Structure of an Intrinsically Disordered Protein: The p53-TAZ2 Case.

Disordered regions and Intrinsically Disordered Proteins (IDPs) are involved in critical cellular processes and may acquire a stable three-dimensional structure only upon binding to their partners. IDPs may follow a folding-after-binding process, known as induced folding, or a folding-before-binding process, known as conformational selection. The transcription factor p53 is involved in the regulation of cellular events that arise upon stress or DNA damage. The p53 domain structure is composed of an N-terminal transactivation domain (p53TAD), a DNA Binding Domain and a tetramerization domain. The activity of TAD is tightly regulated by interactions with cofactors, inhibitors and phosphorylation. To initiate transcription, p53TAD binds to the TAZ2 domain of CBP, a co-transcription factor, and undergoes a folding and binding process, as revealed by the recent NMR structure of the complex. The activity of p53 is regulated by phosphorylation at multiple sites on the TAD domain and recent studies have shown that modifications at three residues affect the binding towards TAZ2. However, we still do not know how these phosphorylations affect the structure of the bound state and, therefore, how they regulate the p53 function. In this work, we have used computational simulations to understand how phosphorylation affects the structure of the p53TAD:TAZ2 complex and regulates the recognition mechanism. Phosphorylation has been proposed to enhance binding by direct interaction with the folded protein or by changing the unbound conformation of IDPs, for example by pre-folding the protein favoring the recognition mechanism. Here, we show an interesting turn in the p53 case: phosphorylation mainly affects the bound structure of p53TAD, highlighting the complexity of IDP protein-protein interactions. Our results are in agreement with previous experimental studies, allowing a clear picture of how p53 is regulated by phosphorylation and giving new insights into how post-translational modifications can regulate the function of IDPs.

Draft Genome Sequence of the Polyextremophilic Halorubrum sp. Strain AJ67, Isolated from Hyperarsenic Lakes in the Argentinian Puna.

Halorubrum sp. strain AJ67, an extreme halophilic UV-resistant archaeon, was isolated from Laguna Antofalla in the Argentinian Puna. The draft genome sequence suggests the presence of potent enzyme candidates that are essential for survival under multiple environmental extreme conditions, such as high UV radiation, elevated salinity, and the presence of critical arsenic concentrations.