The ribosomal RNA processing 1B:protein phosphatase 1 holoenzyme reveals non-canonical PP1 interaction motifs.

The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of substrates by associating with >200 regulatory proteins to form specific holoenzymes. The major PP1 targeting protein in the nucleolus is RRP1B (ribosomal RNA processing 1B). In addition to selectively recruiting PP1ß/PP1γ to the nucleolus, RRP1B also has a key role in ribosome biogenesis, among other functions. How RRP1B binds PP1 and regulates nucleolar phosphorylation signaling is not yet known. Here, we show that RRP1B recruits PP1 via established (RVxF/SILK/ΦΦ) and non-canonical motifs. These atypical interaction sites, the PP1ß/γ specificity, and N-terminal AF-binding pockets rely on hydrophobic interactions that contribute to binding and, via phosphorylation, regulate complex formation. This work advances our understanding of PP1 isoform selectivity, reveals key roles of N-terminal PP1 residues in regulator binding, and suggests that additional PP1 interaction sites have yet to be identified, all of which are necessary for a systems biology understanding of PP1 function.

ASPP proteins discriminate between PP1 catalytic subunits through their SH3 domain and the PP1 C-tail.

Serine/threonine phosphatases such as PP1 lack substrate specificity and associate with a large array of targeting subunits to achieve the requisite selectivity. The tumour suppressor ASPP (apoptosis-stimulating protein of p53) proteins associate with PP1 catalytic subunits and are implicated in multiple functions from transcriptional regulation to cell junction remodelling. Here we show that Drosophila ASPP is part of a multiprotein PP1 complex and that PP1 association is necessary for several in vivo functions of Drosophila ASPP. We solve the crystal structure of the human ASPP2/PP1 complex and show that ASPP2 recruits PP1 using both its canonical RVxF motif, which binds the PP1 catalytic domain, and its SH3 domain, which engages the PP1 C-terminal tail. The ASPP2 SH3 domain can discriminate between PP1 isoforms using an acidic specificity pocket in the n-Src domain, providing an exquisite mechanism where multiple motifs are used combinatorially to tune binding affinity to PP1.

KNL1 Binding to PP1 and Microtubules Is Mutually Exclusive.

The kinetochore scaffold 1 (KNL1) protein coordinates the spindle assembly checkpoint (SAC), a signaling pathway that delays chromosome segregation until all sister chromatids are properly attached to spindle microtubules. Recently, microtubules and protein phosphatase 1 (PP1), which both bind the N-terminal domain of KNL1, have emerged as regulators of the SAC; however, how these proteins interact to contribute to SAC signaling is unknown. Here, we use X-ray crystallography, nuclear magnetic resonance spectroscopy, and biochemical assays to show how KNL1 binds both PP1 and microtubules. Unexpectedly, we discovered that PP1 and microtubules bind KNL1 via overlapping binding sites. Further, we showed that Aurora B kinase phosphorylation results in distinct patterns of KNL1 complex disruption. Finally, combining this data with co-sedimentation assays unequivocally demonstrated that microtubules and PP1 binding to KNL1 is mutually exclusive, with preferential formation of the KNL1:PP1 holoenzyme in the presence of PP1.

A peculiar IclR family transcription factor regulates para-hydroxybenzoate catabolism in Streptomyces coelicolor.

In Streptomyces coelicolor, we identified a para-hydroxybenzoate (PHB) hydroxylase, encoded by gene pobA (SCO3084), which is responsible for conversion of PHB into PCA (protocatechuic acid), a substrate of the ß-ketoadipate pathway which yields intermediates of the Krebs cycle. We also found that the transcription of pobA is induced by PHB and is negatively regulated by the product of SCO3209, which we named PobR. The product of this gene is highly unusual in that it is the apparent fusion of two IclR family transcription factors. Bioinformatic analyses, in vivo transcriptional assays, electrophoretic mobility shift assays (EMSAs), DNase I footprinting, and isothermal calorimetry (ITC) were used to elucidate the regulatory mechanism of PobR. We found that PobR loses its high affinity for DNA (i.e., the pobA operator) in the presence of PHB, the inducer of pobA transcription. PHB binds to PobR with a KD of 5.8 µM. Size-exclusion chromatography revealed that PobR is a dimer in the absence of PHB and a monomer in the presence of PHB. The crystal structure of PobR in complex with PHB showed that only one of the two IclR ligand binding domains was occupied, and defined how the N-terminal ligand binding domain engages the effector ligand.

Expanding the PP2A Interactome by Defining a B56-Specific SLiM.

Specific interactions between proteins govern essential physiological processes including signaling. Many enzymes, especially the family of serine/threonine phosphatases (PSPs: PP1, PP2A, and PP2B/calcineurin/CN), recruit substrates and regulatory proteins by binding short linear motifs (SLiMs), short sequences found within intrinsically disordered regions that mediate specific protein-protein interactions. While tremendous progress had been made in identifying where and how SLiMs bind PSPs, especially PP1 and CN, essentially nothing is known about how SLiMs bind PP2A, a validated cancer drug target. Here we describe three structures of a PP2A-SLiM interaction (B56:pS-RepoMan, B56:pS-BubR1, and B56:pSpS-BubR1), show that this PP2A-specific SLiM is defined as LSPIxE, and then use these data to discover scores of likely PP2A regulators and substrates. Together, these data provide a powerful approach not only for dissecting PP2A interaction networks in cells but also for targeting PP2A diseases, such as cancer.

Interaction of the intermembrane space domain of Tim23 protein with mitochondrial membranes.

Tim23 mediates protein translocation into mitochondria. Although inserted into the inner membrane, the dynamic association of its intermembrane space (IMS) domain with the outer membrane promotes protein import. However, little is known about the molecular basis of this interaction. Here, we demonstrate that the IMS domain of Tim23 tightly associates with both inner and outer mitochondrial membrane-like membranes through a hydrophobic anchor at its N terminus. The structure of membrane-bound Tim23(IMS) is highly dynamic, allowing recognition of both the incoming presequence and other translocase components at the translocation contact. Cardiolipin enhances Tim23 membrane attachment, suggesting that cardiolipin can influence preprotein import.

Molecular basis of the dynamic structure of the TIM23 complex in the mitochondrial intermembrane space.

The presequence translocase TIM23 is a highly dynamic complex in which its subunits can adopt multiple conformations and undergo association-dissociation to facilitate import of proteins into mitochondria. Despite the importance of protein-protein interactions in TIM23, little is known about the molecular details of these processes. Using nuclear magnetic resonance spectroscopy, we characterized the dynamic interaction network of the intermembrane space domains of Tim23, Tim21, Tim50, and Tom22 at single-residue level. We show that Tim23(IMS) contains multiple sites to efficiently interact with the intermembrane space domain of Tim21 and to bind to Tim21, Tim50, and Tom22. In addition, we reveal the atomic details of the dynamic Tim23(IMS)-Tim21(IMS) complex. The combined data support a central role of the intermembrane space domain of Tim23 in the formation and regulation of the presequence translocase.

The intermembrane space domain of Tim23 is intrinsically disordered with a distinct binding region for presequences.

Proteins targeted to the mitochondrial matrix are translocated through the outer and the inner mitochondrial membranes by two protein complexes, the translocase of the outer membrane (TOM) and one of the translocases of the inner membrane (TIM23). The protein Tim23, the core component of TIM23, consists of an N-terminal, soluble domain in the intermembrane space (IMS) and a C-terminal domain that forms the import pore across the inner membrane. Before translocation proceeds, precursor proteins are recognized by the N-terminal domain of Tim23, Tim23N (residues 1-96). By using NMR spectroscopy, we show that Tim23N is a monomeric protein belonging to the family of intrinsically disordered proteins. Titrations of Tim23N with two presequences revealed a distinct binding region of Tim23N formed by residues 71-84. In a charge-hydropathy plot containing all soluble domains of TOM and TIM23, Tim23N was found to be the only domain with more than 40 residues in the IMS that is predicted to be intrinsically disordered, suggesting that Tim23N might function as hub in the mitochondrial import machinery protein network.