The binding selectivity of the C-terminal SH3 domain of Grb2, but not its folding pathway, is dictated by its contiguous SH2 domain.

The adaptor protein Grb2, or growth factor receptor-bound protein 2, possesses a pivotal role in the transmission of fundamental molecular signals in the cell. Despite lacking enzymatic activity, Grb2 functions as a dynamic assembly platform, orchestrating intracellular signals through its modular structure. This study delves into the energetic communication of Grb2 domains, focusing on the folding and binding properties of the C-SH3 domain linked to its neighboring SH2 domain. Surprisingly, while the folding and stability of C-SH3 remain robust and unaffected by SH2 presence, significant differences emerge in the binding properties when considered within the tandem context compared with isolated C-SH3. Through a double mutant cycle analysis, we highlighted a subset of residues, located at the interface with the SH2 domain and far from the binding site, finely regulating the binding of a peptide mimicking a physiological ligand of the C-SH3 domain. Our results have mechanistic implications about the mechanisms of specificity of the C-SH3 domain, indicating that the presence of the SH2 domain optimizes binding to its physiological target, and emphasizing the general importance of considering supramodular multidomain protein structures to understand the functional intricacies of protein-protein interaction domains.

Understanding the molecular basis of folding cooperativity through a comparative analysis of a multidomain protein and its isolated domains.

Although cooperativity is a well-established and general property of folding, our current understanding of this feature in multidomain folding is still relatively limited. In fact, there are contrasting results indicating that the constituent domains of a multidomain protein may either fold independently on each other or exhibit interdependent supradomain phenomena. To address this issue, here we present the comparative analysis of the folding of a tandem repeat protein, comprising two contiguous PDZ domains, in comparison to that of its isolated constituent domains. By analyzing in detail the equilibrium and kinetics of folding at different experimental conditions, we demonstrate that despite each of the PDZ domains in isolation being capable of independent folding, at variance with previously characterized PDZ tandem repeats, the full-length construct folds and unfolds as a single cooperative unit. By exploiting quantitatively, the comparison of the folding of the tandem repeat to those observed for its constituent domains, as well as by characterizing a truncated variant lacking a short autoinhibitory segment, we successfully rationalize the molecular basis of the observed cooperativity and attempt to infer some general conclusions for multidomain systems.

High-Dose Inhaled Nitric Oxide in Acute Hypoxemic Respiratory Failure Due to COVID-19: A Multicenter Phase II Trial.

Rationale: The effects of high-dose inhaled nitric oxide on hypoxemia in coronavirus disease (COVID-19) acute respiratory failure are unknown. Objectives: The primary outcome was the change in arterial oxygenation (PaO2/FiO2) at 48 hours. The secondary outcomes included: time to reach a PaO2/FiO2.300mmHg for at least 24 hours, the proportion of participants with a PaO2/FiO2.300mmHg at 28 days, and survival at 28 and at 90 days. Methods: Mechanically ventilated adults with COVID-19 pneumonia were enrolled in a phase II, multicenter, single-blind, randomized controlled parallel-arm trial. Participants in the intervention arm received inhaled nitric oxide at 80 ppm for 48 hours, compared with the control group receiving usual care (without placebo). Measurements and Main Results: A total of 193 participants were included in the modified intention-to-treat analysis. The mean change in PaO2/FiO2 ratio at 48 hours was 28.3mmHg in the intervention group and 21.4mmHg in the control group (mean difference, 39.1mmHg; 95% credible interval [CrI], 18.1 to 60.3). The mean time to reach a PaO2/FiO2.300mmHg in the interventional group was 8.7 days, compared with 8.4 days for the control group (mean difference, 0.44; 95% CrI, 23.63 to 4.53). At 28 days, the proportion of participants attaining a PaO2/FiO2.300mmHg was 27.7% in the inhaled nitric oxide group and 17.2% in the control subjects (risk ratio, 2.03; 95% CrI, 1.11 to 3.86). Duration of ventilation and mortality at 28 and 90 days did not differ. No serious adverse events were reported. Conclusions: The use of high-dose inhaled nitric oxide resulted in an improvement of PaO2/FiO2 at 48 hours compared with usual care in adults with acute hypoxemic respiratory failure due to COVID-19.

Folding and Binding Kinetics of the Tandem of SH2 Domains from SHP2.

The SH2 domains of SHP2 play a crucial role in determining the function of the SHP2 protein. While the folding and binding properties of the isolated NSH2 and CSH2 domains have been extensively studied, there is limited information about the tandem SH2 domains. This study aims to elucidate the folding and binding kinetics of the NSH2-CSH2 tandem domains of SHP2 through rapid kinetic experiments, complementing existing data on the isolated domains. The results indicate that while the domains generally fold and unfold independently, acidic pH conditions induce complex scenarios involving the formation of a misfolded intermediate. Furthermore, a comparison of the binding kinetics of isolated NSH2 and CSH2 domains with the NSH2-CSH2 tandem domains, using peptides that mimic specific portions of Gab2, suggests a dynamic interplay between NSH2 and CSH2 in binding Gab2 that modulate the microscopic association rate constant of the binding reaction. These findings, discussed in the context of previous research on the NSH2 and CSH2 domains, enhance our understanding of the function of the SH2 domain tandem of SHP2.

Characterization of the folding and binding properties of the PTB domain of FRS2 with phosphorylated and unphosphorylated ligands.

PTB (PhosphoTyrosine Binding) domains are protein domains that exert their function by binding phosphotyrosine residues on other proteins. They are commonly found in a variety of signaling proteins and are important for mediating protein-protein interactions in numerous cellular processes. PTB domains can also exhibit binding to unphosphorylated ligands, suggesting that they have additional binding specificities beyond phosphotyrosine recognition. Structural studies have reported that the PTB domain from FRS2 possesses this peculiar feature, allowing it to interact with both phosphorylated and unphosphorylated ligands, such as TrkB and FGFR1, through different topologies and orientations. In an effort to elucidate the dynamic and functional properties of these protein-protein interactions, we provide a complete characterization of the folding mechanism of the PTB domain of FRS2 and the binding process to peptides mimicking specific regions of TrkB and FGFR1. By analyzing the equilibrium and kinetics of PTB folding, we propose a mechanism implying the presence of an intermediate along the folding pathway. Kinetic binding experiments performed at different ionic strengths highlighted the electrostatic nature of the interaction with both peptides. The specific role of single amino acids in early and late events of binding was pinpointed by site-directed mutagenesis. These results are discussed in light of previous experimental works on these protein systems.

Development of nitric oxide generators to produce high-dose nitric oxide for inhalation therapy.

BACKGROUND: Several nitric oxide (NO) generating devices have been developed to deliver NO between 1 part per million (ppm) and 80 ppm. Although inhalation of high-dose NO may exert antimicrobial effects, the feasibility and safety of producing high-dose (more than 100 ppm) NO remains to be established. In the current study, we designed, developed, and tested three high-dose NO generating devices. METHODS: We constructed three NO generating devices: a double spark plug NO generator, a high-pressure single spark plug NO generator, and a gliding arc NO generator. The NO and NO2 concentrations were measured at different gas flows and under various atmospheric pressures. The double spark plug NO generator was designed to deliver gas through an oxygenator and mixing with pure oxygen. The high-pressure and gliding arc NO generators were used to deliver gas through a ventilator into artificial lungs to mimic delivering high-dose NO in the clinical settings. The energy consumption was measured and compared among the three NO generators. RESULTS: The double spark plug NO generator produced 200 ± 2 ppm (mean ± SD) of NO at gas flow of 8 L/min (or 320 ± 3 ppm at gas flow of 5 L/min) with electrode gap of 3 mm. The nitrogen dioxide (NO2) levels were below 3.0 ± 0.1 ppm when mixing with various volumes of pure oxygen. The addition of a second generator increased the delivered NO from 80 (with one spark plug) to 200 ppm. With the high-pressure chamber, the NO concentration reached 407 ± 3 ppm with continuous air flow at 5 L/min when employing the 3 mm electrode gap under 2.0 atmospheric pressure (ATA). When compared to 1 ATA, NO production was increased 22% at 1.5 ATA and 34% at 2 ATA. The NO level was 180 ± 1 ppm when connecting the device to a ventilator with a constant inspiratory airflow of 15 L/min, and NO2 levels were below 1 (0.93 ± 0.02) ppm. The gliding arc NO generator produced up to 180 ± 4 ppm of NO when connecting the device to a ventilator, and the NO2 level was below 1 (0.91 ± 0.02) ppm in all testing conditions. The gliding arc device required more power (in watts) to generate the same concentrations of NO when compared to double spark plug or high-pressure NO generators. CONCLUSIONS: Our results demonstrated that it is feasible to enhance NO production (more than 100 ppm) while maintaining NO2 level relatively low (less than 3 ppm) with the three recently developed NO generating devices. Future studies might include these novel designs to deliver high doses of inhaled NO as an antimicrobial used to treat upper and lower respiratory tract infections.

Targeting the Grb2 cSH3 domain: Design, synthesis and biological evaluation of the first series of modulators.

Growth factor receptor bound protein 2 (Grb2) is an adaptor protein featured by a nSH3-SH2-cSH3 domains. Grb2 finely regulates important cellular pathways such as growth, proliferation and metabolism and a minor lapse of this tight control may totally change the entire pathway to the oncogenic. Indeed, Grb2 is found overexpressed in many tumours type. Consequently, Grb2 is an attractive therapeutic target for the development of new anticancer drug. Herein, we reported the synthesis and the biological evaluation of a series of Grb2 inhibitors, developed starting from a hit-compound already reported by this research unit. The newly synthesized compounds were evaluated by kinetic binding experiments, and the most promising derivatives were assayed in a short panel of cancer cells. Five of the newly synthesized derivatives proved to be able to bind the targeted protein with valuable inhibitory concentration in one-digit micromolar concentration. The most active compound of this series, derivative 12, showed an inhibitory concentration of about 6 µM for glioblastoma and ovarian cancer cells, and an IC50 of 1.67 for lung cancer cell. For derivative 12, the metabolic stability and the ROS production was also evaluated. The biological data together with the docking studies led to rationalize an early structure activity relationship.

Supertertiary protein structure affects an allosteric network.

The notion that protein function is allosterically regulated by structural or dynamic changes in proteins has been extensively investigated in several protein domains in isolation. In particular, PDZ domains have represented a paradigm for these studies, despite providing conflicting results. Furthermore, it is still unknown how the association between protein domains in supramodules, consitituting so-called supertertiary structures, affects allosteric networks. Here, we experimentally mapped the allosteric network in a PDZ:ligand complex, both in isolation and in the context of a supramodular structure, and show that allosteric networks in a PDZ domain are highly dependent on the supertertiary structure in which they are present. This striking sensitivity of allosteric networks to the presence of adjacent protein domains is likely a common property of supertertiary structures in proteins. Our findings have general implications for prediction of allosteric networks from primary and tertiary structures and for quantitative descriptions of allostery.

Hidden kinetic traps in multidomain folding highlight the presence of a misfolded but functionally competent intermediate.

Although more than 75% of the proteome is composed of multidomain proteins, current knowledge of protein folding is based primarily on studies of isolated domains. In this work, we describe the folding mechanism of a multidomain tandem construct comprising two distinct covalently bound PDZ domains belonging to a protein called Whirlin, a scaffolding protein of the hearing apparatus. In particular, via a synergy between NMR and kinetic experiments, we demonstrate the presence of a misfolded intermediate that competes with productive folding. In agreement with the view that tandem domain swapping is a potential source of transient misfolding, we demonstrate that such a kinetic trap retains native-like functional activity, as shown by the preserved ability to bind its physiological ligand. Thus, despite the general knowledge that protein misfolding is intimately associated with dysfunction and diseases, we provide a direct example of a functionally competent misfolded state. Remarkably, a bioinformatics analysis of the amino acidic sequence of Whirlin from different species suggests that the tendency to perform tandem domain swapping between PDZ1 and PDZ2 is highly conserved, as demonstrated by their unexpectedly high sequence identity. On the basis of these observations, we discuss on a possible physiological role of such misfolded intermediate.

Biophysical Characterization of the Binding Mechanism between the MATH Domain of SPOP and Its Physiological Partners.

SPOP (Speckle-type POZ protein) is an E3 ubiquitin ligase adaptor protein that mediates the ubiquitination of several substrates. Furthermore, SPOP is responsible for the regulation of both degradable and nondegradable polyubiquitination of a number of substrates with diverse biological functions. The recognition of SPOP and its physiological partners is mediated by two protein-protein interaction domains. Among them, the MATH domain recognizes different substrates, and it is critical for orchestrating diverse cellular pathways, being mutated in several human diseases. Despite its importance, the mechanism by which the MATH domain recognizes its physiological partners has escaped a detailed experimental characterization. In this work, we present a characterization of the binding mechanism of the MATH domain of SPOP with three peptides mimicking the phosphatase Puc, the chromatin component MacroH2A, and the dual-specificity phosphatase PTEN. Furthermore, by taking advantage of site-directed mutagenesis, we address the role of some key residues of MATH in the binding process. Our findings are briefly discussed in the context of previously existing data on the MATH domain.

Addressing the Binding Mechanism of the Meprin and TRAF-C Homology Domain of the Speckle-Type POZ Protein Using Protein Engineering.

Protein-protein interactions play crucial roles in a wide range of biological processes, including metabolic pathways, cell cycle progression, signal transduction, and the proteasomal system. For PPIs to fulfill their biological functions, they require the specific recognition of a multitude of interacting partners. In many cases, however, protein-protein interaction domains are capable of binding different partners in the intracellular environment, but they require precise regulation of the binding events in order to exert their function properly and avoid misregulation of important molecular pathways. In this work, we focused on the MATH domain of the E3 Ligase adaptor protein SPOP in order to decipher the molecular features underlying its interaction with two different peptides that mimic its physiological partners: Puc and MacroH2A. By employing stopped-flow kinetic binding experiments, together with extensive site-directed mutagenesis, we addressed the roles of specific residues, some of which, although far from the binding site, govern these transient interactions. Our findings are compatible with a scenario in which the binding of the MATH domain with its substrate is characterized by a fine energetic network that regulates its interactions with different ligands. Results are briefly discussed in the context of previously existing work regarding the MATH domain.

Three Logistic Predictive Models for the Prediction of Mortality and Major Pulmonary Complications after Cardiac Surgery.

Background and Objectives: Pulmonary complications are a leading cause of morbidity after cardiac surgery. The aim of this study was to develop models to predict postoperative lung dysfunction and mortality. Materials and Methods: This was a single-center, observational, retrospective study. We retrospectively analyzed the data of 11,285 adult patients who underwent all types of cardiac surgery from 2003 to 2015. We developed logistic predictive models for in-hospital mortality, postoperative pulmonary complications occurring in the intensive care unit, and postoperative non-invasive mechanical ventilation when clinically indicated. Results: In the "preoperative model" predictors for mortality were advanced age (p < 0.001), New York Heart Association (NYHA) class (p < 0.001) and emergent surgery (p = 0.036); predictors for non-invasive mechanical ventilation were advanced age (p < 0.001), low ejection fraction (p = 0.023), higher body mass index (p < 0.001) and preoperative renal failure (p = 0.043); predictors for postoperative pulmonary complications were preoperative chronic obstructive pulmonary disease (p = 0.007), preoperative kidney injury (p < 0.001) and NYHA class (p = 0.033). In the "surgery model" predictors for mortality were intraoperative inotropes (p = 0.003) and intraoperative intra-aortic balloon pump (p < 0.001), which also predicted the incidence of postoperative pulmonary complications. There were no specific variables in the surgery model predicting the use of non-invasive mechanical ventilation. In the "intensive care unit model", predictors for mortality were postoperative kidney injury (p < 0.001), tracheostomy (p < 0.001), inotropes (p = 0.029) and PaO2/FiO2 ratio at discharge (p = 0.028); predictors for non-invasive mechanical ventilation were kidney injury (p < 0.001), inotropes (p < 0.001), blood transfusions (p < 0.001) and PaO2/FiO2 ratio at the discharge (p < 0.001). Conclusions: In this retrospective study, we identified the preoperative, intraoperative and postoperative characteristics associated with mortality and complications following cardiac surgery.

The cooperative folding of annexin A2 relies on a transient nonnative intermediate.

Annexins (Anxs) are a family of highly homologous proteins that bind and aggregate lipid vesicles in the presence of calcium. All members of the family contain a variable N-terminus determining specific functions, followed by a conserved core region responsible for the general calcium-dependent lipid-binding property. The core structure consists of four homologous domains (DI-DIV), each consisting of a right-handed super-helix of five α-helices. We present data from a combination of site-directed mutagenesis, NMR, and circular dichroism showing that the G25-D34 region of the N-terminus as well as the contacts between residues D38A, R63A, and Q67A of AnxA2-DI are crucial for the autonomous folding and stability of DI of AnxA2. However, we also show that the folding of the full-length protein is very robust in that mutations and truncations that disrupted the folding of AnxA2-DI did not abolish the folding of full-length AnxA2, only lowering its thermal stability. This robustness of the folding of full-length AnxA2 is likely to be mediated by the existence of at least one transient nonnative intermediate as suggested by our kinetic data using stopped-flow fluorescence experiments. We also show that hydrophobic amino acids in AnxA2-DI involved in interfacial contacts with AnxA2-DIV are important for the cooperative folding and stability of the full-length protein. Mutating all of the V57E-V98R-G101Y residues in AnxA2-DI did not affect the folding of the domain, only its stability, but prevented the cooperative folding of the full-length protein. Our collective results favor a highly cooperative and robust folding process mediated by alternative intermediate steps. Since AnxA2 is a multifunctional protein involved in several steps of the progression of cell transformation, these data on structure and folding pathways are therefore crucial to designing anticancer drugs targeting AnxA2.

Fuzziness and Frustration in the Energy Landscape of Protein Folding, Function, and Assembly.

Are all protein interactions fully optimized? Do suboptimal interactions compromise specificity? What is the functional impact of frustration? Why does evolution not optimize some contacts? Proteins and their complexes are best described as ensembles of states populating an energy landscape. These ensembles vary in breadth from narrow ensembles clustered around a single average X-ray structure to broader ensembles encompassing a few different functional "taxonomic" states on to near continua of rapidly interconverting conformations, which are called "fuzzy" or even "intrinsically disordered". Here we aim to provide a comprehensive framework for confronting the structural and dynamical continuum of protein assemblies by combining the concepts of energetic frustration and interaction fuzziness. The diversity of the protein structural ensemble arises from the frustrated conflicts between the interactions that create the energy landscape. When frustration is minimal after folding, it results in a narrow ensemble, but residual frustrated interactions result in fuzzy ensembles, and this fuzziness allows a versatile repertoire of biological interactions. Here we discuss how fuzziness and frustration play off each other as proteins fold and assemble, viewing their significance from energetic, functional, and evolutionary perspectives.We demonstrate, in particular, that the common physical origin of both concepts is related to the ruggedness of the energy landscapes, intramolecular in the case of frustration and intermolecular in the case of fuzziness. Within this framework, we show that alternative sets of suboptimal contacts may encode specificity without achieving a single structural optimum. Thus, we demonstrate that structured complexes may not be optimized, and energetic frustration is realized via different sets of contacts leading to multiplicity of specific complexes. Furthermore, we propose that these suboptimal, frustrated, or fuzzy interactions are under evolutionary selection and expand the biological repertoire by providing a multiplicity of biological activities. In accord, we show that non-native interactions in folding or interaction landscapes can cooperate to generate diverse functional states, which are essential to facilitate adaptation to different cellular conditions. Thus, we propose that not fully optimized structures may actually be beneficial for biological activities of proteins via an alternative set of suboptimal interactions. The importance of such variability has not been recognized across different areas of biology.This account provides a modern view on folding, function, and assembly across the protein universe. The physical framework presented here is applicable to the structure and dynamics continuum of proteins and opens up new perspectives for drug design involving not fully structured, highly dynamic protein assemblies.

Exploring the effect of tethered domains on the folding of Grb2 protein.

Two thirds of eukaryotic proteins have evolved as multidomain constructs, and in vivo, domains fold within a polypeptide chain, with inter-domain interactions possibly crucial for correct folding. However, to date, most of the experimental folding studies are based on domains in isolation. In an effort to better understand multidomain folding, in this work we analyzed, through equilibrium and kinetic folding experiments, the folding properties of the Growth factor receptor-bound protein 2 (Grb2), composed by one SRC homology 2 domain flanked by two SRC homology 3 domains. In particular we compared the kinetic features of the multidomain construct with the domains expressed in isolation. By performing single and double mixing folding experiments, we demonstrated that the folding of the SH2 domain is kinetically trapped in a misfolded intermediate when tethered to the C-SH3. Importantly, within the multidomain construct, misfolding occurred independently if refolding is started with C-SH3 in its unfolded or native state. Interestingly, our data reported a peculiar scenario, in which SH2 and C-SH3 domain reciprocally and transiently interact during folding. Altogether, the analysis of kinetic folding data provided a quantitative description of the multidomain folding of Grb2 protein, discussed under the light of previous works on multidomain folding.

Therapeutic Gases and Inhaled Anesthetics as Adjunctive Therapies in Critically Ill Patients.

The administration of exogenous oxygen to support adequate gas exchange is the cornerstone of respiratory care. In the past few years, other gaseous molecules have been introduced in clinical practice to treat the wide variety of physiological derangement seen in critical care patients.Inhaled nitric oxide (NO) is used for its unique selective pulmonary vasodilator effect. Recent studies showed that NO plays a pivotal role in regulating ischemia-reperfusion injury and it has antibacterial and antiviral activity.Helium, due to its low density, is used in patients with upper airway obstruction and lower airway obstruction to facilitate gas flow and to reduce work of breathing.Carbon monoxide (CO) is a poisonous gas that acts as a signaling molecule involved in many biologic pathways. CO's anti-inflammatory and antiproliferative effects are under investigation in the setting of acute respiratory distress and idiopathic pulmonary fibrosis.Inhaled anesthetics are widely used in the operative room setting and, with the development of anesthetic reflectors, are now a valid option for sedation management in the intensive care unit.Many other gases such as xenon, argon, and hydrogen sulfide are under investigation for their neuroprotective and cardioprotective effects in post-cardiac arrest syndrome.With all these therapeutic options available, the clinician must have a clear understanding of the physiologic basis, therapeutic potential, and possible adverse events of these therapeutic gases. In this review, we will present the therapeutic gases other than oxygen used in clinical practice and we will describe other promising therapeutic gases that are in the early phases of investigation.

SH2 Domains: Folding, Binding and Therapeutical Approaches.

SH2 (Src Homology 2) domains are among the best characterized and most studied protein-protein interaction (PPIs) modules able to bind and recognize sequences presenting a phosphorylated tyrosine. This post-translational modification is a key regulator of a plethora of physiological and molecular pathways in the eukaryotic cell, so SH2 domains possess a fundamental role in cell signaling. Consequently, several pathologies arise from the dysregulation of such SH2-domains mediated PPIs. In this review, we recapitulate the current knowledge about the structural, folding stability, and binding properties of SH2 domains and their roles in molecular pathways and pathogenesis. Moreover, we focus attention on the different strategies employed to modulate/inhibit SH2 domains binding. Altogether, the information gathered points to evidence that pharmacological interest in SH2 domains is highly strategic to developing new therapeutics. Moreover, a deeper understanding of the molecular determinants of the thermodynamic stability as well as of the binding properties of SH2 domains appears to be fundamental in order to improve the possibility of preventing their dysregulated interactions.

How Fast Is Protein-Ligand Association?

There is increasing interest in studying protein interactions and their role in cell biology using kinetics. However, there is confusion about the proper terminology in terms of the distinction between rates and rate constants. We recommend a more stringent use of the words speed, fast, slow, rate, and rate constant.

Functional interplay between protein domains in a supramodular structure involving the postsynaptic density protein PSD-95.

Cell scaffolding and signaling are governed by protein-protein interactions. Although a particular interaction is often defined by two specific domains binding to each other, this interaction often occurs in the context of other domains in multidomain proteins. How such adjacent domains form supertertiary structures and modulate protein-protein interactions has only recently been addressed and is incompletely understood. The postsynaptic density protein PSD-95 contains a three-domain supramodule, denoted PSG, which consists of PDZ, Src homology 3 (SH3), and guanylate kinase-like domains. The PDZ domain binds to the C terminus of its proposed natural ligand, CXXC repeat-containing interactor of PDZ3 domain (CRIPT), and results from previous experiments using only the isolated PDZ domain are consistent with the simplest scenario for a protein-protein interaction; namely, a two-state mechanism. Here we analyzed the binding kinetics of the PSG supramodule with CRIPT. We show that PSG binds CRIPT via a more complex mechanism involving two conformational states interconverting on the second timescale. Both conformational states bound a CRIPT peptide with similar affinities but with different rates, and the distribution of the two conformational states was slightly shifted upon CRIPT binding. Our results are consistent with recent structural findings of conformational changes in PSD-95 and demonstrate how conformational transitions in supertertiary structures can shape the ligand-binding energy landscape and modulate protein-protein interactions.

Templated folding of intrinsically disordered proteins.

Much of our current knowledge of biological chemistry is founded in the structure-function relationship, whereby sequence determines structure that determines function. Thus, the discovery that a large fraction of the proteome is intrinsically disordered, while being functional, has revolutionized our understanding of proteins and raised new and interesting questions. Many intrinsically disordered proteins (IDPs) have been determined to undergo a disorder-to-order transition when recognizing their physiological partners, suggesting that their mechanisms of folding are intrinsically different from those observed in globular proteins. However, IDPs also follow some of the classic paradigms established for globular proteins, pointing to important similarities in their behavior. In this review, we compare and contrast the folding mechanisms of globular proteins with the emerging features of binding-induced folding of intrinsically disordered proteins. Specifically, whereas disorder-to-order transitions of intrinsically disordered proteins appear to follow rules of globular protein folding, such as the cooperative nature of the reaction, their folding pathways are remarkably more malleable, due to the heterogeneous nature of their folding nuclei, as probed by analysis of linear free-energy relationship plots. These insights have led to a new model for the disorder-to-order transition in IDPs termed "templated folding," whereby the binding partner dictates distinct structural transitions en route to product, while ensuring a cooperative folding.