PDZ2-conjugated-PLGA nanoparticles are tiny heroes in the battle against SARS-CoV-2.

The COVID-19 pandemic caused by SARS-CoV-2 has highlighted the urgent need for innovative antiviral strategies to fight viral infections. Although a substantial part of the overall effort has been directed at the Spike protein to create an effective global vaccination strategy, other proteins have also been examined and identified as possible therapeutic targets. Among them, although initially underestimated, there is the SARS-CoV-2 E-protein, which turned out to be a key factor in viral pathogenesis due to its role in virus budding, assembly and spreading. The C-terminus of E-protein contains a PDZ-binding motif (PBM) that plays a key role in SARS-CoV-2 virulence as it is recognized and bound by the PDZ2 domain of the human tight junction protein ZO-1. The binding between the PDZ2 domain of ZO-1 and the C-terminal portion of SARS-CoV-2 E-protein has been extensively characterized. Our results prompted us to develop a possible adjuvant therapeutic strategy aimed at slowing down or inhibiting virus-mediated pathogenesis. Such innovation consists in the design and synthesis of externally PDZ2-ZO1 functionalized PLGA-based nanoparticles to be used as intracellular decoy. Contrary to conventional strategies, this innovative approach aims to capitalize on the E protein-PDZ2 interaction to prevent virus assembly and replication. In fact, the conjugation of the PDZ2 domain to polymeric nanoparticles increases the affinity toward the E protein effectively creating a "molecular sponge" able to sequester E proteins within the intracellular environment of infected cells. Our in vitro studies on selected cellular models, show that these nanodevices significantly reduce SARS-CoV-2-mediated virulence, emphasizing the importance of exploiting viral-host interactions for therapeutic benefit.

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.

The Mechanism of Folding of Human Frataxin in Comparison to the Yeast Homologue - Broad Energy Barriers and the General Properties of the Transition State.

The funneled energy landscape theory suggests that the folding pathway of homologous proteins should converge at the late stages of folding. In this respect, proteins displaying a broad energy landscape for folding are particularly instructive, allowing inferring both the early, intermediate and late stages of folding. In this paper we explore the folding mechanisms of human frataxin, an essential mitochondrial protein linked to the neurodegenerative disorder Friedreich's ataxia. Building upon previous studies on the yeast homologue, the folding pathway of human frataxin is thoroughly examined, revealing a mechanism implying the presence of a broad energy barrier, reminiscent of the yeast counterpart. Through an extensive site-directed mutagenesis, we employed a Φ -value analysis to map native-like contacts in the folding transition state. The presence of a broad energy barrier facilitated the exploration of such contacts in both early and late folding events. We compared results from yeast and human frataxin providing insights into the impact of native topology on the folding mechanism and elucidating the properties of the underlying free energy landscape. The findings are discussed in the context of the funneled energy landscape theory of protein folding.

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.

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.

An intramolecular energetic network regulates ligand recognition in a SH2 domain.

In an effort to investigate the molecular determinants of ligand recognition of the C-terminal SH2 domain of the SHP2 protein, we conducted extensive site-directed mutagenesis and kinetic binding experiments with a peptide mimicking a specific portion of a physiological ligand (the scaffold protein Gab2). Obtained data provided an in-depth characterization of the binding reaction, allowing us to pinpoint residues topologically far from the binding pocket of the SH2 domain to have a role in the recognition and binding of the peptide. The presence of a sparse energetic network regulating the interaction with Gab2 was identified and characterized through double mutant cycle analysis, performed by challenging all the designed site-directed variants of C-SH2 with a Gab2 peptide mutated at +3 position relative to its phosphorylated tyrosine, a key residue for C-SH2 binding specificity. Results highlighted non-optimized residues involved in the energetic network regulating the binding with Gab2, which may be at the basis of the ability of this SH2 domain to interact with different partners in the intracellular environment. Moreover, a detailed analysis of kinetic and thermodynamic parameters revealed the role of the residue at +3 position on Gab2 in the early and late events of the binding reaction with the C-SH2 domain.

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.

Monomeric and dimeric states of human ZO1-PDZ2 are functional partners of the SARS-CoV-2 E protein.

The Envelope (E) protein of SARS-CoV-2 plays a key role in virus maturation, assembly, and virulence mechanisms. The E protein is characterized by the presence of a PDZ-binding motif (PBM) at its C-terminus that allows it to interact with several PDZ-containing proteins in the intracellular environment. One of the main binding partners of the SARS-CoV-2 E protein is the PDZ2 domain of ZO1, a protein with a crucial role in the formation of epithelial and endothelial tight junctions (TJs). In this work, through a combination of analytical ultracentrifugation analysis and equilibrium and kinetic folding experiments, we show that ZO1-PDZ2 domain is able to fold in a monomeric state, an alternative form to the dimeric conformation that is reported to be functional in the cell for TJs assembly. Importantly, surface plasmon resonance (SPR) data indicate that the PDZ2 monomer is fully functional and capable of binding the C-terminal portion of the E protein of SARS-CoV-2, with a measured affinity in the micromolar range. Moreover, we present a detailed computational analysis of the complex between the C-terminal portion of E protein with ZO1-PDZ2, both in its monomeric conformation (computed as a high confidence AlphaFold2 model) and dimeric conformation (obtained from the Protein Data Bank), by using both polarizable and nonpolarizable simulations. Together, our results indicate both the monomeric and dimeric states of PDZ2 to be functional partners of the E protein, with similar binding mechanisms, and provide mechanistic and structural information about a fundamental interaction required for the replication of SARS-CoV-2.

Different electrostatic forces drive the binding kinetics of SARS-CoV, SARS-CoV-2 and MERS-CoV Envelope proteins with the PDZ2 domain of ZO1.

The Envelope protein (E) is a structural protein encoded by the genome of SARS-CoV, SARS-CoV-2 and MERS-CoV Coronaviruses. It is poorly present in the virus but highly expressed in the host cell, with prominent role in virus assembly and virulence. The E protein possesses a PDZ-binding motif (PBM) at its C terminus that allows it to interact with host PDZ domain containing proteins. ZO1 is a key protein in assembling the cytoplasmic plaque of epithelial and endothelial Tight Junctions (TJs) as well as in determining cell differentiation, proliferation and polarity. The PDZ2 domain of ZO1 is known to interact with the Coronaviruses Envelope proteins, however the molecular details of such interaction have not been established. In this paper we directly measured, through Fluorescence Resonance Energy Transfer and Stopped-Flow methodology, the binding kinetics of the PDZ2 domain of ZO1 with peptides mimicking the C-terminal portion of the Envelope protein from SARS-CoV, SARS-CoV-2 and MERS-CoV in different ionic strength conditions. Interestingly, the peptide mimicking the E protein from MERS-CoV display much higher microscopic association rate constant with PDZ2 compared to SARS-CoV and SARS-CoV-2 suggesting a stronger contribution of electrostatic forces in the early events of binding. A comparison of thermodynamic and kinetic data obtained at increasing ionic strengths put in evidence different contribution of electrostatics in the recognition and complex formation events for the three peptides. Our data are discussed under the light of available structural data of PDZ2 domain of ZO1 and of previous works about these protein systems.

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.

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.

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.

Cryptic binding properties of a transient folding intermediate in a PDZ tandem repeat.

PDZ domains are the most diffused protein-protein interaction modules of the human proteome and are often present in tandem repeats. An example is PDZD2, a protein characterized by the presence of six PDZ domains that undergoes a proteolytic cleavage producing sPDZD2, comprising a tandem of two PDZ domains, namely PDZ5 and PDZ6. Albeit the physiopathological importance of sPDZD2 is well-established, the interaction with endogenous ligands has been poorly characterized. To understand the determinants of the stability and function of sPDZD2, we investigated its folding pathway. Our data highlights the presence of a complex scenario involving a transiently populated folding intermediate that may be accumulated from the concurrent denaturation of both PDZ5 and PDZ6 domains. Importantly, double jump kinetic experiments allowed us to pinpoint the ability of this transient intermediate to bind the physiological ligand of sPDZD2 with increased affinity compared to the native state. In summary, our results provide an interesting example of a functionally competent misfolded intermediate, which may exert a cryptic function that is not captured from the analysis of the native state only.

Folding and Binding Mechanisms of the SH2 Domain from Crkl.

SH2 domains are structural modules specialized in the recognition and binding of target sequences containing a phosphorylated tyrosine residue. They are mostly incorporated in the 3D structure of scaffolding proteins that represent fundamental regulators of several signaling pathways. Among those, Crkl plays key roles in cell physiology by mediating signals from a wide range of stimuli, and its overexpression is associated with several types of cancers. In myeloid cells expressing the oncogene BCR/ABL, one interactor of Crkl-SH2 is the focal adhesion protein Paxillin, and this interaction is crucial in leukemic transformation. In this work, we analyze both the folding pathway of Crkl-SH2 and its binding reaction with a peptide mimicking Paxillin, under different ionic strength and pH conditions, by using means of fluorescence spectroscopy. From a folding perspective, we demonstrate the presence of an intermediate along the reaction. Moreover, we underline the importance of the electrostatic interactions in the early event of recognition, occurring between the phosphorylated tyrosine of the Paxillin peptide and the charge residues of Crkl-SH2. Finally, we highlight a pivotal role of a highly conserved histidine residue in the stabilization of the binding complex. The experimental results are discussed in light of previous works on other SH2 domains.

Characterization of early and late transition states of the folding pathway of a SH2 domain.

Albeit SH2 domains are abundant protein-protein interaction modules with fundamental roles in the regulation of several physiological and molecular pathways in the cell, the available information about the determinants of their thermodynamic stability and folding properties are still very limited. In this work, we provide a quantitative characterization of the folding pathway of the C-terminal SH2 domain of SHP2, conducted through a combination of site-directed mutagenesis and kinetic (un)folding experiments (Φ-value analysis). The energetic profile of the folding reaction of the C-SH2 domain is described by a three-state mechanism characterized by the presence of two transition states and a high-energy intermediate. The production of 29 site-directed variants allowed us to calculate the degree of native-like interactions occurring in the early and late events of the folding reaction. Data analysis highlights the presence of a hydrophobic folding nucleus surrounded by a lower degree of structure in the early events of folding, further consolidated as the reaction proceeds towards the native state. Interestingly, residues physically located in the functional region of the domain reported unusual Φ-values, a hallmark of the presence of transient misfolding. We compared our results with previous ones obtained for the N-terminal SH2 domain of SHP2. Notably, a conserved complex folding mechanism implying the presence of a folding intermediate arise from comparison, and the relative stability of such intermediate appears to be highly sequence dependent. Data are discussed under the light of previous works on SH2 domains.

On the Effects of Disordered Tails, Supertertiary Structure and Quinary Interactions on the Folding and Function of Protein Domains.

The vast majority of our current knowledge about the biochemical and biophysical properties of proteins derives from in vitro studies conducted on isolated globular domains. However, a very large fraction of the proteins expressed in the eukaryotic cell are structurally more complex. In particular, the discovery that up to 40% of the eukaryotic proteins are intrinsically disordered, or possess intrinsically disordered regions, and are highly dynamic entities lacking a well-defined three-dimensional structure, revolutionized the structure-function paradigm and our understanding of proteins. Moreover, proteins are mostly characterized by the presence of multiple domains, influencing each other by intramolecular interactions. Furthermore, proteins exert their function in a crowded intracellular milieu, transiently interacting with a myriad of other macromolecules. In this review we summarize the literature tackling these themes from both the theoretical and experimental perspectives, highlighting the effects on protein folding and function that are played by (i) flanking disordered tails; (ii) contiguous protein domains; (iii) interactions with the cellular environment, defined as quinary structures. We show that, in many cases, both the folding and function of protein domains is remarkably perturbed by the presence of these interactions, pinpointing the importance to increase the level of complexity of the experimental work and to extend the efforts to characterize protein domains in more complex contexts.

Probing the Effects of Local Frustration in the Folding of a Multidomain Protein.

Our current knowledge of protein folding is primarily based on experimental data obtained from isolated domains. In fact, because of their complexity, multidomain proteins have been elusive to the experimental characterization. Thus, the folding of a domain in isolation is generally assumed to resemble what should be observed for more complex structural architectures. Here we compared the folding mechanism of a protein domain in isolation and in the context of its supramodular multidomain structure. By carrying out an extensive mutational analysis we illustrate that while the early events of folding are malleable and influenced by the absence/presence of the neighboring structures, the late events appear to be more robust. These effects may be explained by analyzing the local frustration patterns of the domain, providing critical support for the funneled energy landscape theory of protein folding, and highlighting the role of protein frustration in sculpting the early events of the reaction.