Investigating Homology between Proteins using Energetic Profiles

Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding) and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved local stability, may provide guidance for a future thermodynamically informed classification of protein homology

The regulatory subunit of PKA-I remains partially structured and undergoes β-aggregation upon thermal denaturation

Background: The regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B. Methodology/Principal Findings: As revealed by circular dichroism (CD) and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212-216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers. Conclusions/Significance: Thermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation. © 2011 Dao et al

Polycation-π Interactions Are a Driving Force for Molecular Recognition by an Intrinsically Disordered Oncoprotein Family

Molecular recognition by intrinsically disordered proteins (IDPs) commonly involves specific localized contacts and target-induced disorder to order transitions. However, some IDPs remain disordered in the bound state, a phenomenon coined "fuzziness", often characterized by IDP polyvalency, sequence-insensitivity and a dynamic ensemble of disordered bound-state conformations. Besides the above general features, specific biophysical models for fuzzy interactions are mostly lacking. The transcriptional activation domain of the Ewing's Sarcoma oncoprotein family (EAD) is an IDP that exhibits many features of fuzziness, with multiple EAD aromatic side chains driving molecular recognition. Considering the prevalent role of cation-π interactions at various protein-protein interfaces, we hypothesized that EAD-target binding involves polycation- π contacts between a disordered EAD and basic residues on the target. Herein we evaluated the polycation-π hypothesis via functional and theoretical interrogation of EAD variants. The experimental effects of a range of EAD sequence variations, including aromatic number, aromatic density and charge perturbations, all support the cation-π model. Moreover, the activity trends observed are well captured by a coarse-grained EAD chain model and a corresponding analytical model based on interaction between EAD aromatics and surface cations of a generic globular target. EAD-target binding, in the context of pathological Ewing's Sarcoma oncoproteins, is thus seen to be driven by a balance between EAD conformational entropy and favorable EAD-target cation-π contacts. Such a highly versatile mode of molecular recognition offers a general conceptual framework for promiscuous target recognition by polyvalent IDPs. © 2013 Song et al

Protonation States of Remote Residues Affect Binding-Release Dynamics of the Ligand but not the Conformation of apo Ferric Binding Protein

We have studied the apo (Fe3+ free) form of periplasmic ferric binding protein (FbpA) under different conditions and we have monitored the changes in the binding and release dynamics of H2PO4- that acts as a synergistic anion in the presence of Fe3+. Our simulations predict a dissociation constant of 2.2$\pm$0.2 mM which is in remarkable agreement with the experimentally measured value of 2.3$\pm$0.3 mM under the same ionization strength and pH conditions. We apply perturbations relevant for changes in environmental conditions as (i) different values of ionic strength (IS), and (ii) protonation of a group of residues to mimic a different pH environment. Local perturbations are also studied by protonation or mutation of a site distal to the binding region that is known to mechanically manipulate the hinge-like motions of FbpA. We find that while the average conformation of the protein is intact in all simulations, the H2PO4- dynamics may be substantially altered by the changing conditions. In particular, the bound fraction which is 20$\%$ for the wild type system is increased to 50$\%$ with a D52A mutation/protonation and further to over 90$\%$ at the protonation conditions mimicking those at pH 5.5. The change in the dynamics is traced to the altered electrostatic distribution on the surface of the protein which in turn affects hydrogen bonding patterns at the active site. The observations are quantified by rigorous free energy calculations. Our results lend clues as to how the environment versus single residue perturbations may be utilized for regulation of binding modes in hFbpA systems in the absence of conformational changes.Comment: 26 pages, 4 figure

Using least median of squares for structural superposition of flexible proteins

<p>Abstract</p> <p>Background</p> <p>The conventional superposition methods use an ordinary least squares (LS) fit for structural comparison of two different conformations of the same protein. The main problem of the LS fit that it is sensitive to outliers, i.e. large displacements of the original structures superimposed.</p> <p>Results</p> <p>To overcome this problem, we present a new algorithm to overlap two protein conformations by their atomic coordinates using a robust statistics technique: least median of squares (LMS). In order to effectively approximate the LMS optimization, the forward search technique is utilized. Our algorithm can automatically detect and superimpose the rigid core regions of two conformations with small or large displacements. In contrast, most existing superposition techniques strongly depend on the initial LS estimating for the entire atom sets of proteins. They may fail on structural superposition of two conformations with large displacements. The presented LMS fit can be considered as an alternative and complementary tool for structural superposition.</p> <p>Conclusion</p> <p>The proposed algorithm is robust and does not require any prior knowledge of the flexible regions. Furthermore, we show that the LMS fit can be extended to multiple level superposition between two conformations with several rigid domains. Our fit tool has produced successful superpositions when applied to proteins for which two conformations are known. The binary executable program for Windows platform, tested examples, and database are available from <url>https://engineering.purdue.edu/PRECISE/LMSfit</url>.</p

Drug export and allosteric coupling in a multidrug transporter revealed by molecular simulations

Multidrug resistance is a serious problem in current chemotherapy. The efflux system largely responsible for resistance in Escherichia coli contains the drug transporter, AcrB. The structures of AcrB were solved in 2002 as the symmetric homo-trimer, and then in 2006 as the asymmetric homo-trimer. The latter suggested a functionally rotating mechanism. Here, by molecular simulations of the AcrB porter domain, we uncovered allosteric coupling and the drug export mechanism in the AcrB trimer. Allosteric coupling stabilized the asymmetric structure with one drug molecule bound, which validated the modelling. Drug dissociation caused a conformational change and stabilized the symmetric structure, providing a unified view of the structures reported in 2002 and 2006. A dynamic study suggested that, among the three potential driving processes, only protonation of the drug-bound protomer can drive the functional rotation and simultaneously export the drug

Prediction of native-state hydrogen exchange from perfectly funneled energy landscapes

Simulations based on perfectly funneled energy landscapes often capture many of the kinetic features of protein folding. We examined whether simulations based on funneled energy functions can also describe fluctuations in native-state protein ensembles. We quantitatively compared the site-specific local stability determined from structure-based folding simulations, with hydrogen exchange protection factors measured experimentally for ubiquitin, chymotrypsin inhibitor 2, and staphylococcal nuclease. Different structural definitions for the open and closed states based on the number of native contacts for each residue, as well as the hydrogen-bonding state, or a combination of both criteria were evaluated. The predicted exchange patterns agree with the experiments under native conditions, indicating that protein topology indeed has a dominant effect on the exchange kinetics. Insights into the simplest mechanistic interpretation of the amide exchange process were thus obtained.Fil: Craig, Patricio Oliver. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. University of California San Diego. Department of Chemistry and Biochemistry; Estados UnidosFil: Lätzer, Joachim. Rutgers University. BioMaPS Institute; Estados UnidosFil: Weinkam, Patrick. University of California at San Francisco. Department of Bioengineering and Therapeutic Sciences; Estados UnidosFil: Hoffman, Ryan M. B.. University Of California At San Diego; Estados UnidosFil: Ferreiro, Diego. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Komives, Elizabeth A.. University Of California At San Diego; Estados UnidosFil: Wolynes, Peter G.. University Of California At San Diego; Estados Unido

Peptide Conformer Acidity Analysis of Protein Flexibility Monitored by Hydrogen Exchange†

ABSTRACT: The amide hydrogens that are exposed to solvent in the high-resolution X-ray structures of ubiquitin, FK506-binding protein, chymotrypsin inhibitor 2, and rubredoxin span a billion-fold range in hydroxide-catalyzed exchange rates which are predictable by continuum dielectric methods. To facilitate analysis of transiently accessible amides, the hydroxide-catalyzed rate constants for every backbone amide of ubiquitin were determined under near physiological conditions. With the previously reported NMR-restrained molecular dynamics ensembles of ubiquitin (PDB codes 2NR2 and 2K39) used as representations of the Boltzmann-weighted conformational distribution, nearly all of the exchange rates for the highly exposed amides were more accurately predicted than by use of the high-resolution X-ray structure. More strikingly, predictions for the amide hydrogens of the NMR relaxation-restrained ensemble that become exposed to solvent in more than one but less than half of the 144 protein conformations in this ensemble were almost as accurate. In marked contrast, the exchange rates for many of the analogous amides in the residual dipolar coupling-restrained ubiquitin ensemble are substantially overestimated, as was particularly evident for the Ile 44 to Lys 48 segment which constitutes the primary interaction site for the proteasome targeting enzymes involved in polyubiquitylation. For both ensembles, “excited state ” conformers in this active site region having markedly elevated peptide acidities are represented at a population level that is 102 to 103 abov