The Conformational Plasticity Vista of PDZ Domains

Part of this researchwas funded by the Andalusian Regional Government (http://www.juntadeandalucia.es) [grant number CVI-5915], and the author currently acknowledges funds from a reincorporation contract from the University of Granada.The PDZ domain (PSD95-Discs large-ZO1) is a widespread modular domain present in the living organisms. Aprevalent function in the PDZ family is to serve as sca olding and adaptor proteins connecting multiple partners in signaling pathways. An explanation of the flexible functionality in this domain family, based just on a static perspective of the structure–activity relationship, might fall short. More dynamic and conformational aspects in the protein fold can be the reasons for such functionality. Folding studies indeed showed an ample and malleable folding landscape for PDZ domains where multiple intermediate states were experimentally detected. Allosteric phenomena that resemble energetic coupling between residues have also been found in PDZ domains. Additionally, several PDZ domains are modulated by post-translational modifications, which introduce conformational switches that affect binding. Altogether, the ability to connect diverse partners might arise from the intrinsic plasticity of the PDZ fold.Andalusian Regional Government CVI-5915University of Granad

Common features in the unfolding and misfolding of PDZ domains and beyond: the modulatory effect of domain swapping and extra-elements

PDZ domains are protein-protein interaction modules sharing the same structural arrangement. To discern whether they display common features in their unfolding/misfolding behaviour we have analyzed in this work the unfolding thermodynamics, together with the misfolding kinetics, of the PDZ fold using three archetypical examples: the second and third PDZ domains of the PSD95 protein and the Erbin PDZ domain. Results showed that all domains passed through a common intermediate, which populated upon unfolding, and that this in turn drove the misfolding towards worm-like fibrillar structures. Thus, the unfolding/misfolding behaviour appears to be shared within these domains. We have also analyzed how this landscape can be modified upon the inclusion of extra-elements, as it is in the nNOS PDZ domain, or the organization of swapped species, as happens in the second PDZ domain of the ZO2 protein. Although the intermediates still formed upon thermal unfolding, the misfolding was prevented to varying degrees

The Impact of Extra-Domain Structures and Post-Translational Modifications in the Folding/Misfolding Behaviour of the Third PDZ Domain of MAGUK Neuronal Protein PSD-95

The modulation of binding affinities and specificities by post-translational modifications located out from the binding pocket of the third PDZ domain of PSD-95 (PDZ3) has been reported recently. It is achieved through an intra-domain electrostatic network involving some charged residues in the β2–β3 loop (were a succinimide modification occurs), the α3 helix (an extra-structural element that links the PDZ3 domain with the following SH3 domain in PSD-95, and contains the phosphorylation target Tyr397), and the ligand peptide. Here, we have investigated the main structural and thermodynamic aspects that these structural elements and their related post-translational modifications display in the folding/misfolding pathway of PDZ3 by means of site-directed mutagenesis combined with calorimetry and spectroscopy. We have found that, although all the assayed mutations generate proteins more prone to aggregation than the wild-type PDZ3, those directly affecting the α3 helix, like the E401R substitution or the truncation of the whole α3 helix, increase the population of the DSC-detected intermediate state and the misfolding kinetics, by organizing the supramacromolecular structures at the expense of the two β-sheets present in the PDZ3 fold. However, those mutations affecting the β2–β3 loop, included into the prone-to-aggregation region composed by a single β-sheet comprising β2 to β4 chains, stabilize the trimeric intermediate previously shown in the wild-type PDZ3 and slow-down aggregation, also making it partly reversible. These results strongly suggest that the α3 helix protects to some extent the PDZ3 domain core from misfolding. This might well constitute the first example where an extra-element, intended to link the PDZ3 domain to the following SH3 in PSD-95 and in other members of the MAGUK family, not only regulates the binding abilities of this domain but it also protects PDZ3 from misfolding and aggregation. The influence of the post-translational modifications in this regulatory mechanism is also discussed.This research was supported by grants CVI-05915, from the Andalusian Regional Government; BIO2009-13261-C02 and BIO2012-39922-C02, from the Spanish Ministry of Science and Education and FEDER; PI13-01330 from Instituto de Salud Carlos III and SGR09-0761 from the Generalitat de Catalunya. J.M-C. received a postdoctoral contract from the Spanish Ministry of Science and Education. M.M-A. was supported by a PIF (UAB) fellowship

A Panel of TrpB Biocatalysts Derived from Tryptophan Synthase through the Transfer of Mutations that Mimic Allosteric Activation

Naturally occurring enzyme homologues often display highly divergent activity with non-natural substrates. Exploiting this diversity with enzymes engineered for new or altered function, however, is laborious because the engineering must be replicated for each homologue. A small set of mutations of the tryptophan synthase β-subunit (TrpB) from Pyrococcus furiosus, which mimics the activation afforded by binding of the α-subunit, was demonstrated to have a similar activating effect in different TrpB homologues with as little as 57 % sequence identity. Kinetic and spectroscopic analyses indicate that the mutations function through the same mechanism: mimicry of α-subunit binding. From these enzymes, we identified a new TrpB catalyst that displays a remarkably broad activity profile in the synthesis of 5-substituted tryptophans. This demonstrates that allosteric activation can be recapitulated throughout a protein family to explore natural sequence diversity for desirable biocatalytic transformations

Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope

Unlocking reactivity of TrpB: a general biocatalytic platform for synthesis of tryptophan analogs

Derivatives of the amino acid tryptophan (Trp) serve as precursors for the chemical and biological synthesis of complex molecules with a wide range of biological properties. Trp analogues are also valuable as building blocks for medicinal chemistry and as tools for chemical biology. While the enantioselective synthesis of Trp analogues is often lengthy and requires the use of protecting groups, enzymes have the potential to synthesize such products in fewer steps and with the pristine chemo- and stereoselectivity that is a hallmark of biocatalysis. The enzyme TrpB is especially attractive because it can form Trp analogues directly from serine (Ser) and the corresponding indole analogue. However, many potentially useful substrates, including bulky or electron-deficient indoles, are poorly accepted. We have applied directed evolution to TrpB from Pyrococcus furiosus and Thermotoga maritima to generate a suite of catalysts for the synthesis of previously intractable Trp analogues. For the most challenging substrates, such as nitroindoles, the key to improving activity lay in the mutation of a universally conserved and mechanistically important residue, E104. The new catalysts express at high levels (>200 mg/L of Escherichia coli culture) and can be purified by heat treatment; they can operate up to 75 °C (where solubility is enhanced) and can synthesize enantiopure Trp analogues substituted at the 4-, 5-, 6-, and 7-positions, using Ser and readily available indole analogues as starting materials. Spectroscopic analysis shows that many of the activating mutations suppress the decomposition of the active electrophilic intermediate, an amino-acrylate, which aids in unlocking the synthetic potential of TrpB

Post-Translational Modifications Modulate Ligand Recognition by the Third PDZ Domain of the MAGUK Protein PSD-95

The relative promiscuity of hub proteins such as postsynaptic density protein-95 (PSD-95) can be achieved by alternative splicing, allosteric regulation, and post-translational modifications, the latter of which is the most efficient method of accelerating cellular responses to environmental changes in vivo. Here, a mutational approach was used to determine the impact of phosphorylation and succinimidation post-translational modifications on the binding affinity of the postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ3) domain of PSD-95. Molecular dynamics simulations revealed that the binding affinity of this domain is influenced by an interplay between salt-bridges linking the α3 helix, the β2–β3 loop and the positively charged Lys residues in its high-affinity hexapeptide ligand KKETAV. The α3 helix is an extra structural element that is not present in other PDZ domains, which links PDZ3 with the following SH3 domain in the PSD-95 protein. This regulatory mechanism was confirmed experimentally via thermodynamic and NMR chemical shift perturbation analyses, discarding intra-domain long-range effects. Taken together, the results presented here reveal the molecular basis of the regulatory role of the α3 extra-element and the effects of post-translational modifications of PDZ3 on its binding affinity, both energetically and dynamically.This research was supported by grants CVI-05915, from the Andalusian Regional Government (http://www.juntadeandalucia.es), BIO2009-13261-C02 and BIO2012-39922-C02, from the Spanish Ministry of Science and Innovation (http://www.idi.mineco.gob.es/portal/site​/MICINN/) and FEDER. JMC received a postdoctoral contract from the Spanish Ministry of Science and Innovation. CCV was a recipient of a Formación de Personal Investigador fellowship from the Spanish Ministry of Science and Innovation

Tryptophan synthase uses an atypical mechanism to achieve substrate specificity

Tryptophan synthase (TrpS) catalyzes the final steps in the biosynthesis of L-tryptophan from L-serine (Ser) and indole-3-glycerol phosphate (IGP). We report that native TrpS can also catalyze a productive reaction with L-threonine (Thr), leading to (2S,3S)-β-methyltryptophan. Surprisingly, β-substitution occurs in vitro with a 3.4-fold higher catalytic efficiency for Ser over Thr using saturating indole, despite >82,000-fold preference for Ser in direct competition using IGP. Structural data identify a novel product binding site and kinetic experiments clarify the atypical mechanism of specificity: Thr binds efficiently but decreases the affinity for indole and disrupts the allosteric signaling that regulates the catalytic cycle

Phage display identification of nanomolar ligands for human NEDD4-WW3: Energetic and dynamic implications for the development of broad-spectrum antivirals

This research has been financed by grants BIO2016-78746-C2-1-R and PID2020-112895RB-100 from the Spanish Ministry of Education and Science (I.L.) including AEI/FEDER EU funds. R.N.H. was funded in part by National Institutes of Health grants AI138052 and AI138630. M. I.B. and J.M.C. were recipients of a research contract from the Spanish Ministry of Education and Science. F.C. was funded by a predoctoral fellowship from the Andalusian Government P10-CVI-5915. J.M.C. ac-knowledges a reincorporation research contract from the University of Granada. We thank Dr. Sachdev Sidhu for his invaluable assistance setting up the phage display techniques in our laboratory. We also thank the support of the C.I.C. of the University of Granada.The recognition of PPxY viral Late domains by the third WW domain of the human HECT-E3 ubiquitin ligase NEDD4 (NEDD4-WW3) is essential for the budding of many viruses. Blocking these interactions is a promising strategy to develop broad-spectrum antivirals. As all WW domains, NEDD4-WW3 is a challenging therapeutic target due to the low binding affinity of its natural interactions, its high conformational plasticity, and its complex thermodynamic behavior. In this work, we set out to investigate whether high affinity can be achieved for monovalent ligands binding to the isolated NEDD4-WW3 domain. We show that a competitive phage-display set-up allows for the identification of high-affinity peptides showing inhibitory activity of viral budding. A detailed biophysical study combining calorimetry, nuclear magnetic resonance, and molecular dynamic simulations reveals that the improvement in binding affinity does not arise from the establishment of new interactions with the domain, but is associated to conformational restrictions imposed by a novel C-terminal -LFP motif in the ligand, unprecedented in the PPxY interactome. These results, which highlight the complexity of WW domain interactions, provide valuable insight into the key elements for high binding affinity, of interest to guide virtual screening campaigns for the identification of novel therapeutics targeting NEDD4-WW3 interactions.Spanish Government BIO2016-78746-C2-1-R PID2020-112895RB-100 AEI/FEDER EU funds AI138052 AI138630United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA P10-CVI-5915German Research Foundation (DFG)University of Granad