Frustration Sculpts the Early Stages of Protein Folding.

The funneled energy landscape theory implies that protein structures are minimally frustrated. Yet, because of the divergent demands between folding and function, regions of frustrated patterns are present at the active site of proteins. To understand the effects of such local frustration in dictating the energy landscape of proteins, here we compare the folding mechanisms of the two alternative spliced forms of a PDZ domain (PDZ2 and PDZ2as) that share a nearly identical sequence and structure, while displaying different frustration patterns. The analysis, based on the kinetic characterization of a large number of site-directed mutants, reveals that although the late stages for folding are very robust and biased by native topology, the early stages are more malleable and dominated by local frustration. The results are briefly discussed in the context of the energy-landscape theory.

Recognition and binding of apocytochrome c to P. aeruginosa CcmI, a component of cytochrome c maturation machinery.

The biogenesis of c-type cytochromes (Cytc) is a process that in Gram-negative bacteria demands the coordinated action of different periplasmic proteins (CcmA-I), whose specific roles are still being investigated. Activities of Ccm proteins span from the chaperoning of heme b in the periplasm to the specific reduction of oxidized apocytochrome (apoCyt) cysteine residues and to chaperoning and recognition of the unfolded apoCyt before covalent attachment of the heme to the cysteine thiols can occur. We present here the functional characterization of the periplasmic domain of CcmI from the pathogen Pseudomonas aeruginosa (Pa-CcmI*). Pa-CcmI* is composed of a TPR domain and a peculiar C-terminal domain. Pa-CcmI* fulfills both the ability to recognize and bind to P. aeruginosa apo-cytochrome c551 (Pa-apoCyt) and a chaperoning activity towards unfolded proteins, as it prevents citrate synthase aggregation in a concentration-dependent manner. Equilibrium and kinetic experiments with Pa-CcmI*, or its isolated domains, with peptides mimicking portions of Pa-apoCyt sequence allow us to quantify the molecular details of the interaction between Pa-apoCyt and Pa-CcmI*. Binding experiments show that the interaction occurs at the level of the TPR domain and that the recognition is mediated mainly by the C-terminal sequence of Pa-apoCyt. The affinity of Pa-CcmI* to full-length Pa-apoCyt or to its C-terminal sequence is in the range expected for a component of a multi-protein complex, whose task is to receive the apoCyt and to deliver it to other components of the apoCyt:heme b ligation protein machinery.

A dual signalling pathway for the hypoxic expression of lipid genes, dependent on the glucose sensor Rag4, is revealed by the analysis of the KlMGA2 gene in Kluyveromyces lactis.

In the respiratory yeast Kluyveromyces lactis, little is known about the factors regulating the metabolic response to oxygen shortage. After searching for homologues of characterized Saccharomyces cerevisiae regulators of the hypoxic response, we identified a gene that we named KlMGA2, which is homologous to MGA2. The deletion of KlMGA2 strongly reduced both the fermentative and respiratory growth rate and altered fatty acid composition and the unsaturation index of membranes. The reciprocal heterologous expression of MGA2 and KlMGA2 in the corresponding deletion mutant strains suggested that Mga2 and KlMga2 are functional homologues. KlMGA2 transcription was induced by hypoxia and the glucose sensor Rag4 mediated the hypoxic induction of KlMGA2. Transcription of lipid biosynthetic genes KlOLE1, KlERG1, KlFAS1 and KlATF1 was induced by hypoxia and was dependent on KlMga2, except for KlOLE1. Rag4 was required for hypoxic induction of transcription for both KlMga2-dependent (KlERG1) and KlMga2-independent (KlOLE1) structural genes.

Regulation of the Human Phosphatase PTPN4 by the inter-domain linker connecting the PDZ and the phosphatase domains.

Human protein tyrosine phosphatase non-receptor type 4 (PTPN4) has been shown to prevent cell death. The active form of human PTPN4 consists of two globular domains, a PDZ (PSD-95/Dlg/ZO-1) domain and a phosphatase domain, tethered by a flexible linker. Targeting its PDZ domain abrogates this protection and triggers apoptosis. We previously demonstrated that the PDZ domain inhibits the phosphatase activity of PTPN4 and that the mere binding of a PDZ ligand is sufficient to release the catalytic inhibition. We demonstrate here that the linker connecting the PDZ domain and the phosphatase domain is involved in the regulation of the phosphatase activity in both PDZ-related inhibition and PDZ ligand-related activation events. We combined bioinformatics and kinetic studies to decipher the role of the linker in the PTPN4 activity. By comparing orthologous sequences, we identified a conserved patch of hydrophobic residues in the linker. We showed that mutations in this patch affect the regulation of the PTPN4 bidomain indicating that the PDZ-PDZ ligand regulation of PTPN4 is a linker-mediated mechanism. However, the mutations do not alter the binding of the PDZ ligand. This study strengthens the notion that inter-domain linker can be of functional importance in enzyme regulation of large multi-domain proteins.

Understanding the effect of alternative splicing in the folding and function of the second PDZ from protein tyrosine phosphatase-BL.

PDZ domains are the most prominent biological structural domains involved in protein-protein interactions in the human cell. The second PDZ domain of the protein tyrosine phosphatase BL (PDZ2) interacts and binds the C-termini of the tumour suppressor protein APC and of the LIM domain-containing protein RIL. One isoform of PDZ2 (PDZ2as) involves an alternative spliced form that exhibits an insertion of 5 residues in a loop. PDZ2as abrogates binding to its partners, even if the insertion is directly located in its binding pocket. Here, we investigate the folding and function of PDZ2as, in comparison to the previously characterized PDZ2 domain. Data reveal that, whilst the thermodynamic stability of PDZ2as appears as nearly identical to that of PDZ2, the insertion of 5 amino acids induces formation of some weak transient non-native interactions in the folding transition state, as mirrored by a concomitant increase of both the folding and unfolding rate constants. From a functional perspective, we show that the decrease in affinity is caused by a pronounced decrease of the association rate constants (by nearly ten fold), with no effect on the microscopic dissociation rate constants. The results are briefly discussed in the context of previous work on PDZ domains.

Demonstration of a folding after binding mechanism in the recognition between the measles virus NTAIL and X domains.

In the past decade, a wealth of experimental data has demonstrated that a large fraction of proteins, while functional, are intrinsically disordered at physiological conditions. Many intrinsically disordered proteins (IDPs) undergo a disorder-to-order transition upon binding to their biological targets, a phenomenon known as induced folding. Induced folding may occur through two extreme mechanisms, namely conformational selection and folding after binding. Although the pre-existence of ordered structures in IDPs is a prerequisite for conformational selection, it does not necessarily commit to this latter mechanism, and kinetic studies are needed to discriminate between the two possible scenarios. So far, relatively few studies have addressed this issue from an experimental perspective. Here, we analyze the interaction kinetics between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the viral phosphoprotein. Data reveal that NTAIL recognizes XD by first forming a weak encounter complex in a disordered conformation, which is subsequently locked-in by a folding step; i.e., binding precedes folding. The implications of our kinetic results, in the context of previously reported equilibrium data, are discussed. These results contribute to enhancing our understanding of the molecular mechanisms by which IDPs recognize their partners and represent a paradigmatic example of the need of kinetic methods to discriminate between reaction mechanisms.

The mechanism of binding of the second PDZ domain from the Protein Tyrosine Phosphatase-BL to the Adenomatous Polyposis Coli tumor suppressor.

Many biological processes are regulated by the interaction between protein domains and their corresponding binding partners. The PDZ domain is one of the most common protein-protein interaction modules in mammalian cells, whose role is to bind C-terminal sequences of specific targets. The second PDZ domain from the Protein Tyrosine Phosphatase-BL (PDZ2) binds to the C-terminal of Adenomatous Polyposis Coli protein (APC), one of the major tumor suppressor whose task is to regulate cell adhesion and proliferation. Here, we present a detailed kinetics analysis of the interaction between PDZ2 domain and a peptide mimicking the PDZ binding motif of APC. By analyzing data obtained at different experimental conditions, we propose a plausible mechanism for binding. Furthermore, a comparison between the dissociation rate constant measured by different methodologies allow us to identify an additional kinetic step, which is likely to arise from a conformational change of PDZ2 occurring after binding. The data are discussed on the light of previous work on PDZ domains.