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

Double Monoubiquitination Modifies the Molecular Recognition Properties of p15PAF Promoting Binding to the Reader Module of Dnmt1

The proliferating cell nuclear antigen (PCNA)-associated factor p15PAF is a nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15PAF gene is overexpressed in several types of human cancer, and its function is regulated by monoubiquitination of two lysines (K15 and K24) at the protein N-terminal region. We have previously shown that p15PAF is an intrinsically disordered protein which partially folds upon binding to PCNA and independently contacts DNA through its Nterminal tail. Here we present an NMR conformational characterization of p15PAF monoubiquitinated at both K15 and K24 via a disulfide bridge mimicking the isopeptide bond. We show that doubly monoubiquitinated p15PAF is monomeric, intrinsically disordered, and binds to PCNA as nonubiquitinated p15PAF does but interacts with DNA with reduced affinity. Our SAXS-derived conformational ensemble of doubly monoubiquitinated p15PAF shows that the ubiquitin moieties, separated by eight disordered residues, form transient dimers because of the high local effective ubiquitin concentration. This observation and the sequence similarity with histone H3 N-terminal tail suggest that doubly monoubiquitinated p15PAF is a binding target of DNA methyl transferase Dnmt1, as confirmed by calorimetry. Therefore, doubly monoubiquitinated p15PAF directly interacts with PCNA and recruits Dnmt1 for maintenance of DNA methylation during replication.Spanish Ministerio de Economía y Competitividad and the Fondo Europeo de Desarrollo Regional (MINECO/FEDER) [CTQ2017-83810-R to F.J.B.]; Labex EpiGenMed, an “Investissements d”avenir’ program [ANR-10-LABX-12-01 to PB]. MOSTMicro [LISBOA-01-0145-FEDER-007660 to T.N.C. and H.M.]. A.G.M. acknowledges Spanish MINECO for predoctoral contract BE-2015-075847, and the CIC bioGUNE acknowledges MINECO for the Severo Ochoa accreditation Sev-2016-0644. The CBS-Montpellier is a member of France-BioImaging (FBI) and the French Infrastructure for Integrated Structural Biology (FRISBI), two national infrastructures supported by the French National Research Agency (ANR-10-INSB-04-01 and ANR-10-INSB- 05, respectively)

The p12 subunit of human polymerase delta uses an atypical PIP box for molecular recognition of proliferating cell nuclear antigen (PCNA)

Human DNA polymerase delta is essential for DNA replication and acts in conjunction with the processivity factor proliferating cell nuclear antigen (PCNA). In addition to its catalytic subunit (p125), pol delta comprises three regulatory subunits (p50, p68, and p12). PCNA interacts with all of these subunits, but only the interaction with p68 has been structurally characterized. Here, we report solution NMR–, isothermal calorimetry–, and X-ray crystallography–based analyses of the p12–PCNA interaction, which takes part in the modulation of the rate and fidelity of DNAsynthesis by pol delta.We show that p12 binds with micromolar affinity to the classical PIP-binding pocket of PCNA via a highly atypical PIP box located at the p12Nterminus. Unlike the canonical PIP box of p68, the PIP box of p12 lacks the conserved glutamine; binds through a 2-fork plug made of an isoleucine and a tyrosine residue at 3 and 8 positions, respectively; and is stabilized by an aspartate at 6 position, which creates a network of intramolecular hydrogen bonds. These findings add to growing evidence that PCNAcan bind a diverse range of protein sequences that may be broadly grouped as PIP-like motifs as has been previously suggested

The p12 subunit of human polymerase δ uses an atypical PIP-box for molecular recognition of proliferating cell nuclear antigen (PCNA).

Human DNA polymerase δ is essential for DNA replication and acts in conjunction with the processivity factor proliferating cell nuclear antigen (PCNA). Besides its catalytic subunit (p125), pol δ comprises three regulatory subunits (p50, p68, and p12). PCNA interacts with all of these subunits, but only the interaction with p68 has been structurally characterized. Here, we report solution NMR-, isothermal titration calorimetry-, and X-ray crystallography-based analyses of the p12-PCNA interaction, which takes part in the modulation of the rate and fidelity of DNA synthesis by pol δ. We show that p12 binds with micromolar affinity to the classical PIP-binding pocket of PCNA via a highly atypical PIP-box located at the p12 N terminus. Unlike the canonical PIP-box of p68, the PIP-box of p12 lacks the conserved glutamine, binds through a 2-fork plug made of an isoleucine and a tyrosine residue at +3 and +8 positions, respectively, and is stabilized by an aspartate at +6 position, which creates a network of intra-molecular hydrogen bonds. These findings add to growing evidence that PCNA can bind a diverse range of protein sequences that may be broadly grouped as PIP-like motifs as has been previously suggested