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The PKC-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains, and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF)-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.

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The protein kinase PDK1 phosphorylates at least 24 distinct substrates, all of which belong to the AGC protein kinase group. Some substrates, such as conventional PKCs, undergo phosphorylation by PDK1 during their synthesis and subsequently get activated by DAG and Calcium. On the other hand, other substrates, including members of the Akt/PKB, S6K, SGK, and RSK families, undergo phosphorylation and activation downstream of PI3-kinase signaling. This review presents two accepted molecular mechanisms that determine the precise and timely phosphorylation of different substrates by PDK1. The first mechanism involves the colocalization of PDK1 with Akt/PKB in the presence of PIP3. The second mechanism involves the regulated docking interaction between the hydrophobic motif (HM) of substrates and the PIF-pocket of PDK1. This interaction, in trans, is equivalent to the molecular mechanism that governs the activity of AGC kinases through their HMs intramolecularly. PDK1 has been instrumental in illustrating the bi-directional allosteric communication between the PIF-pocket and the ATP-binding site and the potential of the system for drug discovery. PDK1's interaction with substrates is not solely regulated by the substrates themselves. Recent research indicates that full-length PDK1 can adopt various conformations based on the positioning of the PH domain relative to the catalytic domain. These distinct conformations of full-length PDK1 can influence the interaction and phosphorylation of substrates. Finally, we critically discuss recent findings proposing that PIP3 can directly regulate the activity of PDK1, which contradicts extensive in vitro and in vivo studies conducted over the years.

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Over the past decade, myriads of studies have highlighted the central role of protein condensation in subcellular compartmentalization and spatiotemporal organization of biological processes. Conceptually, protein condensation stands at the highest level in protein structure hierarchy, accounting for the assembly of bodies ranging from thousands to billions of molecules and for densities ranging from dense liquids to solid materials. In size, protein condensates range from nanocondensates of hundreds of nanometers (mesoscopic clusters) to phase-separated micron-sized condensates. In this review, we focus on protein nanocondensation, a process that can occur in subsaturated solutions and can nucleate dense liquid phases, crystals, amorphous aggregates, and fibers. We discuss the nanocondensation of proteins in the light of general physical principles and examine the biophysical properties of several outstanding examples of nanocondensation. We conclude that protein nanocondensation cannot be fully explained by the conceptual framework of micron-scale biomolecular condensation. The evolution of nanocondensates through changes in density and order is currently under intense investigation, and this should lead to the development of a general theoretical framework, capable of encompassing the full range of sizes and densities found in protein condensates.

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Intrinsically disordered proteins (IDPs) have numerous dynamic conformations. Given the difficulties in tracking temporarily folded states of this kind of protein, methods such as molecular modeling and molecular dynamics (MD) simulations make the process less costly, less laborious, and more detailed. Few plant IDPs have been characterized so far, such as proteins from the Abscisic acid, Stress and Ripening (ASR) family. The present work applied, for the first time, the two above-mentioned tools to test the feasibility of determining a three-dimensional transition model of OsASR5 and to investigate the relationship between OsASR5 and zinc. We found that one of OsASR5's conformers contains α-helices, turns, and loops and that the metal binding resulted in a predominance of α-helix. This stability is possibly imperative for the transcription factor activity. The promoter region of a sugar transporter was chosen to test this hypothesis and free energy calculations showed how the ion is mandatory for this complex formation. The results produced here aim to clarify which conformation the protein in the bound state assumes and which residues are involved in the process, besides developing the understanding of how the flexibility of these proteins can contribute to the response to environmental stresses.

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Protein conformation and cell compartmentalization are fundamental concepts and subjects of vast scientific endeavors. In the last two decades, we have witnessed exciting advances that unveiled the conjunction of these concepts. An avalanche of studies highlighted the central role of biomolecular condensates in membraneless subcellular compartmentalization that permits the spatiotemporal organization and regulation of myriads of simultaneous biochemical reactions and macromolecular interactions. These studies have also shown that biomolecular condensation, driven by multivalent intermolecular interactions, is mediated by order-disorder transitions of protein conformation and by protein domain architecture. Conceptually, protein condensation is a distinct level in protein conformational landscape in which collective folding of large collections of molecules takes place. Biomolecular condensates arise by the physical process of phase separation and comprise a variety of bodies ranging from membraneless organelles to liquid condensates to solid-like conglomerates, spanning lengths from mesoscopic clusters (nanometers) to micrometer-sized objects. In this review, we summarize and discuss recent work on the assembly, composition, conformation, material properties, thermodynamics, regulation, and functions of these bodies. We also review the conceptual framework for future studies on the conformational dynamics of condensed proteins in the regulation of cellular processes.

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The von Willebrand disease (vWD) is the most common hereditary bleeding disorder caused by defects of the von Willebrand Factor (vWF), a large extracellular protein in charge of adhering platelets to sites of vascular lesions. vWF performs this essential homeostatic task via specific protein-protein interactions between the vWF A1 domain and the platelet receptor, the glycoprotein Ib alpha (GPIBα). The two naturally occurring vWF A1 domain mutations G1324A and G1324S, near the GPIBα binding site, induce a dramatic decrease in platelet adhesion, resulting in a bleeding disorder classified as type 2M vWD. However, the reason for the drastic phenotypic response induced by these two supposedly minor modifications remains unclear. We addressed this question using a combination of equilibrium-molecular dynamics (MD) and nonequilibrium MD-based free energy simulations. Our data confirms that both mutations maintain the highly stable Rossmann fold of the vWF A1 domain. G1324A and G1324S mutations hardly changed the per-residue flexibility of the A1 domain but induced a global conformational change affecting the region near the binding site to GPIBα. Furthermore, we observed two significant changes in the vWF A1 domain upon mutation, the global redistribution of the internal mechanical stress and the increased thermodynamic stability of the A1 domain. These observations are consistent with previously reported mutations increasing the melting temperature. Overall, our results support the idea of thermodynamic conformational restriction of A1-before the binding to GPIBα-as a crucial factor determining the loss-of-function of the G1324A(S) vWD mutants.

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INTRODUCTION: Insulin and insulin-like growth factor type 1 (IGF1) regulate multiple physiological functions by acting on the insulin receptor (IR) and insulin-like growth factor type 1 receptor (IGF1R). The insulin analog glargine differs from insulin in three residues (GlyA21, ArgB31, ArgB32), and it is converted to metabolite M1 (lacks residues ArgB31 and ArgB32) by in vivo processing. It is known that activation of these receptors modulates pathways related to metabolism, cell division, and growth. Though, the structures and structural basis of the glargine interaction with these receptors are not known. AIM: To generate predictive structural models, and to analyze the drug/receptor interactions in the system formed by glargine, its metabolite M1, IR, and IGF1R by using bioinformatics tools. METHODS: Ligand/receptor models were built by homology modeling using SWISSMODEL, and surface interactions were analyzed using Discovery Studio® Visualizer. Target and hetero target sequences and appropriate template structures were used for modeling. RESULTS: Our glargine/IR and metabolite M1/IR models showed an overall symmetric T-shaped conformation and full occupancy with four ligand molecules. The glargine/IR model revealed that the glargine residues ArgB31 and ArgB32 fit in a hydrophilic region formed by the α-chain C-terminal helix (αCT) and the cysteine-rich region (CR) domain of this receptor, close to the CR residues Arg270-Arg271-Gln272 and αCT residue Arg717. Regarding IGF1R, homologous ligand/receptor models were further built assuming that the receptor is in a symmetrical T-shaped conformation and is fully occupied with four ligand molecules, similar to what we described for IR. Our glargine/IGF1R model showed the interaction of the glargine residues ArgB31 and ArgB32 with Glu264 and Glu305 in the CR domain of IGF1R. CONCLUSION: Using bioinformatics tools and predictive modeling, our study provides a better understanding of the glargine/receptor interactions.

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Isolated or as a part of multidomain proteins, Sterol Carrier Protein 2 (SCP2) exhibits high affinity and broad specificity for different lipidic and hydrophobic compounds. A wealth of structural information on SCP2 domains in all forms of life is currently available; however, many aspects of its ligand binding activity are poorly understood. ylSCP2 is a well-characterized single domain SCP2 from the yeast Yarrowia lipolytica. Herein, we report the X-ray structure of unliganded ylSCP2 refined to 2.0 Å resolution. Comparison with the previously solved liganded ylSCP2 structure unveiled a novel mechanism for binding site occlusion. The liganded ylSCP2 binding site is a large cavity with a volume of more than 800 Å3. In unliganded ylSCP2 the binding site is reduced to about 140 Å3. The obliteration is caused by a swing movement of the C-terminal α helix 5 and a subtle compaction of helices 2-4. Previous pairwise comparisons were between homologous SCP2 domains with a uncertain binding status. The reported unliganded ylSCP2 structure allows for the first time a fully controlled comparative analysis of the conformational effects of ligand occupation dispelling several doubts regarding the architecture of SCP2 binding site.

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Research within the field of colloidal liquid aphrons (CLAs) for enzyme immobilization has often used ionic surfactants for the retention of enzymes. Although these charged interactions allow for enhanced immobilization, they can often lead to denaturation of enzyme activity, and even release of the protein. Sodium alginate has been used in drug delivery applications due to its low toxicity and charged interactions that allow for encapsulation. Hence, alginate systems can be used as an alternative to ionic surfactants in CLA immobilization. This paper presents, for the first time, the use of sodium alginate as potential ligand for enhanced CLA immobilization. The use of five model proteins; lysozyme, bovine serum albumin, ovalbumin, insulin, and α-chymotrypsin, of various pIs and hydrophobicities, showed the relevance of electrostatic interactions in promoting binding with sodium alginate when the pH < pI, with 100% immobilization attributed to alginate incorporated CLAs over general nonionic formulations. Furthermore, above their pI, >80% protein recovery was observed, with activity and conformation comparable to their native counterparts. Finally, the use of proteolysis showed that as the degree of ionic bonding increased between the protein and sodium alginate, the degree of protease resistance decreased due to conformational changes experienced during binding.

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[Formula: see text]-Lactamases (penicillinases) facilitate bacterial resistance to antibiotics and are excellent theoretical and experimental models in protein structure, dynamics and evolution. Bacillus licheniformis exo-small penicillinase (ESP) is a Class A [Formula: see text]-lactamase with three tryptophan residues located one in each of its two domains and one in the interface between domains. The conformational landscape of three well-characterized ESP Trp[Formula: see text]Phe mutants was characterized in equilibrium unfolding experiments by measuring tryptophan fluorescence, far-UV CD, activity, hydrodynamic radius, and limited proteolysis. The Trp[Formula: see text]Phe substitutions had little impact on the native conformation, but changed the properties of the partially folded states populated at equilibrium. The results were interpreted in the framework of modern theories of protein folding.

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Cinnamic acid (CA) and methyl cinnamate (MC) have attracted interest of researchers because of their broad therapeutic functions. Here, we investigated the interaction of CA and MC with bovine serum albumin (BSA) at pH 3.5, 5.0, and 7.4 using fluorescence spectroscopy, differential scanning nanocalorimetry, and measurements of interfacial tension, size, and zeta potential. BSA formed a complex with the ligands with stoichiometry of approximately 1.0. At pH 7.4, CA-BSA complex formation was entropically driven. The interaction between MC and BSA was more favorable than with CA and was enthalpically driven under the same conditions. The pH played an important role in BSA conformation, which altered the manner in which it interacts with the ligands. Interestingly, both CA and MC had no effect on the surface tension of BSA/air interfaces. These data contribute to the knowledge of CA/MC-BSA interactions and provide important data for application in the food industry.

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A statistical analysis of circa 20,000 X-ray structures evidenced the effects of temperature of data collection on protein intramolecular distances and degree of compaction. Identical chains with data collected at cryogenic ultralow temperatures (≤160K) showed a radius of gyration (Rg) significantly smaller than at moderate temperatures (≥240K). Furthermore, the analysis revealed the existence of structures with a Rg significantly smaller than expected for cryogenic temperatures. In these ultracompact cases, the unusually small Rg could not be specifically attributed to any experimental parameter or crystal features. Ultracompaction involves most atoms and results in their displacement toward the center of the molecule. Ultracompact structures on average have significantly shorter van der Waals and hydrogen bonds than expected for ultralow temperature structures. In addition, the number of van der Waals contacts was larger in ultracompact than in ultralow temperature structures. The structure of these ultracompact states was analyzed in detail and the implication and possible causes of the phenomenon are discussed.

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Circular dichroism (CD) spectroscopy is a fast, powerful, well-established, and widely used analytical technique in the biophysical and structural biology community to study protein secondary structure and to track changes in protein conformation in different environments. The use of the intense light of a synchrotron beam as the light source for collecting CD measurements has emerged as an enhanced method, known as synchrotron radiation circular dichroism (SRCD) spectroscopy, that has several advantages over the conventional CD method, including a significant spectral range extension for data collection, deeper access to the lower limit (cut-off) of conventional CD spectroscopy, an improved signal-to-noise ratio to increase accuracy in the measurements, and the possibility to collect measurements in highly absorbing solutions. In this review, we discuss different applications of the SRCD technique by researchers from Latin America. In this context, we specifically look at the use of this method for examining the secondary structure and conformational behavior of proteins belonging to the four main classes of the hierarchical protein domain classification CATH (Class, Architecture, Topology, Homology) database, focusing on the advantages and improvements associated with SRCD spectroscopy in terms of characterizing proteins composed of different structural elements.

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The unordered secondary structural content of an intrinsically disordered protein (IDP) is susceptible to conformational changes induced by many different external factors, such as the presence of organic solvents, removal of water, changes in temperature, binding to partner molecules, and interaction with lipids and/or other ligands. In order to characterize the high-flexibility nature of an IDP, circular dichroism (CD) spectroscopy is a particularly useful method due to its capability of monitoring both subtle and remarkable changes in different environments, relative ease in obtaining measurements, the small amount of sample required, and the capability for sample recovery (sample not damaged) and others. Using synchrotron radiation as the light source for CD spectroscopy represents the state-of-the-art version of this technique with feasibility of accessing the lower wavelength UV region, and therefore presenting a series of advantages over conventional circular dichroism (cCD) to monitor a protein conformational behavior, check protein stability, detect ligand binding, and many others. In this paper, we have performed a comparative study using cCD and SRCD methods for investigating the secondary structure and the conformational behavior of natively unfolded proteins: MEG-14 and soybean trypsin inhibitor. We show that the SRCD technique greatly improves the analysis and accuracy of the studies on the conformations of IDPs.

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The objectives of this study were to evaluate protein thermal stability, water-protein interaction, microstructure, and protein conformation between PSE-like and normal chicken breast meat batters. Sixty pale, soft, and exudative (PSE)-like (L*>53, pH24 h<5.7) and 60 normal (46

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Δ78Δ is a second generation functional all-ß sheet variant of IFABP (intestinal fatty acid binding protein) corresponding to the fragment 29-106 of the parent protein. This protein and its predecessor, Δ98Δ (segment 29-126 of IFABP), were initially uncovered by controlled proteolysis. Remarkably, although IFABP and Δ98Δ are monomers in solution, Δ78Δ adopts a stable dimeric structure. With the aim of identifying key structural features that modulate the aggregation of ß-proteins, we evaluate here the structure and aggregation propensity of Δ78Δ. The 2,2,2-trifluoroethanol (TFE) induced aggregation of this protein shows a primary nucleation-elongation mechanism, characterized by the stabilization of a dimeric nucleus. Its rate of production from the co-solvent induced aggregation prone state governs the kinetics of polymerization. In this context, the value of Δ78Δ lies in the fact that - being a stable dimeric species - it reduces an otherwise bimolecular reaction to a unimolecular one. Interestingly, even though Δ78Δ and IFABP display similar conformational stability, the abrogated form of IFABP shows an enhanced aggregation rate, revealing the ancillary role played on this process by the free energy of the native proteins. Δ78Δ share with IFABP and Δ98Δ a common putative aggregation-prone central peptide. Differences in the exposure/accessibility of this segment dictated by the environment around this region might underlie the observed variations in the speed of aggregation. Lessons learnt from this natural dimeric protein might shed light on the early conformational events leading to ß-conversion from barrels to amyloid aggregates.

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Septins are filament-forming GTP-binding proteins involved in important cellular events, such as cytokinesis, barrier formation, and membrane remodeling. Here, we present two crystal structures of the GTPase domain of a Schistosoma mansoni septin (SmSEPT10), one bound to GDP and the other to GTP. The structures have been solved at an unprecedented resolution for septins (1.93 and 2.1 Å, respectively), which has allowed for unambiguous structural assignment of regions previously poorly defined. Consequently, we provide a reliable model for functional interpretation and a solid foundation for future structural studies. Upon comparing the two complexes, we observe for the first time the phenomenon of a strand slippage in septins. Such slippage generates a front-back communication mechanism between the G and NC interfaces. These data provide a novel mechanistic framework for the influence of nucleotide binding to the GTPase domain, opening new possibilities for the study of the dynamics of septin filaments.

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The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca(2+) with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. To assess the conformational behavior of the Ca(2+) binding domain, we also studied the occlusion of Ca(2+), both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca(2+) and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only.

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