A Consensus Binding Motif for the PP4 Protein Phosphatase.

Dynamic protein phosphorylation constitutes a fundamental regulatory mechanism in all organisms. Phosphoprotein phosphatase 4 (PP4) is a conserved and essential nuclear serine and threonine phosphatase. Despite the importance of PP4, general principles of substrate selection are unknown, hampering the study of signal regulation by this phosphatase. Here, we identify and thoroughly characterize a general PP4 consensus-binding motif, the FxxP motif. X-ray crystallography studies reveal that FxxP motifs bind to a conserved pocket in the PP4 regulatory subunit PPP4R3. Systems-wide in silico searches integrated with proteomic analysis of PP4 interacting proteins allow us to identify numerous FxxP motifs in proteins controlling a range of fundamental cellular processes. We identify an FxxP motif in the cohesin release factor WAPL and show that this regulates WAPL phosphorylation status and is required for efficient cohesin release. Collectively our work uncovers basic principles of PP4 specificity with broad implications for understanding phosphorylation-mediated signaling in cells.

The NSP3 protein of SARS-CoV-2 binds fragile X mental retardation proteins to disrupt UBAP2L interactions.

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1, FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and reduced levels of viral antigen in lungs during the early stages of infection. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins and provides molecular insight into the possible underlying molecular defects in fragile X syndrome.

Substrate recognition principles for the PP2A-B55 protein phosphatase.

The PP2A-B55 phosphatase regulates a plethora of signaling pathways throughout eukaryotes. How PP2A-B55 selects its substrates presents a severe knowledge gap. By integrating AlphaFold modelling with comprehensive high resolution mutational scanning, we show that α-helices in substrates bind B55 through an evolutionary conserved mechanism. Despite a large diversity in sequence and composition, these α-helices share key amino acid determinants that engage discrete hydrophobic and electrostatic patches. Using deep learning protein design, we generate a specific and potent competitive peptide inhibitor of PP2A-B55 substrate interactions. With this inhibitor, we uncover that PP2A-B55 regulates the nuclear exosome targeting complex by binding to an α-helical recruitment module in RBM7. Collectively, our findings provide a framework for the understanding and interrogation of PP2A-B55 in health and disease.

SARS-CoV-2 hijacks fragile X mental retardation proteins for efficient infection.

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1 and FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and have delayed disease onset in vivo. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins for efficient infection and provides molecular insight to the possible underlying molecular defects in fragile X syndrome.

Identification and Optimization of Novel Small-Molecule Cas9 Inhibitors by Cell-Based High-Throughput Screening.

CRISPR/Cas9 has revolutionized several areas of life science; however, methods to control the Cas9 activity are needed for both scientific and therapeutic applications. Anti-CRISPR proteins are known to inhibit the CRISPR/Cas adaptive immunity; however, in vivo delivery of such proteins is problematic. Instead, small-molecule Cas9 inhibitors could serve as useful tools due to their permeable, proteolytically stable, and non-immunogenic nature. Here, we identified a small-molecule ligand with anti-CRISPR/Cas9 activity through a high-throughput screening utilizing an Escherichia coli selection system. Extensive structure-activity relationship studies, which involved a deconstruction-reconstruction strategy, resulted in a range of analogues with significant improvements in the inhibitory activity. Based on NMR and electrophoretic mobility shift assays, we propose that the inhibitory action of these compounds likely results from direct binding to apo-Cas9, preventing Cas9:gRNA complex formation. These molecules may find use as Cas9 modulators in various applications.

Two functionally distinct kinetochore pools of BubR1 ensure accurate chromosome segregation.

The BubR1/Bub3 complex is an important regulator of chromosome segregation as it facilitates proper kinetochore-microtubule interactions and is also an essential component of the spindle assembly checkpoint (SAC). Whether BubR1/Bub3 localization to kinetochores in human cells stimulates SAC signalling or only contributes to kinetochore-microtubule interactions is debated. Here we show that two distinct pools of BubR1/Bub3 exist at kinetochores and we uncouple these with defined BubR1/Bub3 mutants to address their function. The major kinetochore pool of BubR1/Bub3 is dependent on direct Bub1/Bub3 binding and is required for chromosome alignment but not for the SAC. A distinct pool of BubR1/Bub3 localizes by directly binding to phosphorylated MELT repeats on the outer kinetochore protein KNL1. When we prevent the direct binding of BubR1/Bub3 to KNL1 the checkpoint is weakened because BubR1/Bub3 is not incorporated into checkpoint complexes efficiently. In conclusion, kinetochore localization supports both known functions of BubR1/Bub3.

A left-handed alpha-helix containing both L- and D-amino acids: the solution structure of the antimicrobial lipodepsipeptide tolaasin.

The 18-amino acid cytolytic lipodepsipeptide tolaasin, produced in culture by virulent strains of Pseudomonas tolaasii, is the causal agent of the brown blotch disease of the cultivated mushroom. Tolaasin has a sequence of D-amino acids in its N-terminal region, then alternates L- and D-amino acids, and bears a C-terminal lactone macrocycle composed of 5-residues. The solution structure of tolaasin in sodium dodecyl sulfate was studied by 2D-NMR spectroscopy and molecular dynamics simulated annealing calculations. Tolaasin forms an amphipathic left-handed alpha-helix in the regionDPro2-DalloThr14 comprising the sequence of seven D-amino acids and the adjacent L-D-L-D-D-region. To the best of our knowledge, this is the first recognized example of a left-handed alpha-helix including both D- and L-amino acids. The lactone macrocycle adopts a "boat-like" conformation and is shifted from the helical axis as to form a "golf-club" overall conformation. These structural features will be of importance in understanding, and preventing, tolaasin's role in the bacterial colonization of the host plant, and its toxic action on cells. Furthermore, the observed antimicrobial activity together with the potential resistance to enzymatic degradation and the increased antigenicity (both due to the presence of L- and D-amino acids) strongly suggests for tolaasin a potential role as a template model for the design of new therapeutic antibacterial molecules.

Structural determinants of salmon calcitonin bioactivity: the role of the Leu-based amphipathic alpha-helix.

Salmon calcitonin (sCT) forms an amphipathic helix in the region 9-19, with the C-terminal decapeptide interacting with the helix (Amodeo, P., Motta, A., Strazzullo, G., Castiglione Morelli, M. A. (1999) J. Biomol. NMR 13, 161-174). To uncover the structural requirements for the hormone bioactivity, we investigated several sCT analogs. They were designed so as to alter the length of the central helix by removal and/or replacement of flanking residues and by selectively mutating or deleting residues inside the helix. The helix content was assessed by circular dichroism and NMR spectroscopies; the receptor binding affinity in human breast cancer cell line T 47D and the in vivo hypocalcemic activity were also evaluated. In particular, by NMR spectroscopy and molecular dynamics calculations we studied Leu(23),Ala(24)-sCT in which Pro(23) and Arg(24) were replaced by helix inducing residues. Compared with sCT, it assumes a longer amphipathic alpha-helix, with decreased binding affinity and one-fifth of the hypocalcemic activity, therefore supporting the idea of a relationship between a definite helix length and bioactivity. From the analysis of other sCT mutants, we inferred that the correct helix length is located in the 9-19 region and requires long range interactions and the presence of specific regions of residues within the sequence for high binding affinity and hypocalcemic activity. Taken together, the structural and biological data identify well defined structural parameters of the helix for sCT bioactivity.