MPP OUTSIDE REQUEST FORM Review Date 8/3/11 Requestor(s):Brad Gibson/Liza Pon Institution:Buck Institute, Novato, CA/Columbia University Request Title:mouse and human mitochondrial protein PINK1 Protein NameORF IDSource OrganismHeatTarget Request IDTarget ScorepIResiduesSize (Da) PINK1GO Homo sapiens30TR Pink1Mus musculus Alt. names: Serine/threonine-protein kinase PINK1, mitochondrial | BRPK | PTEN-induced putative kinase protein 1 Requestor notes (edited): Brad Gibson The structure of Pink1 (PTEN induced putative kinase 1 [ Mus musculus ] GeneID: 68943), a mitochondrial kinase associated with early onset Parkinson’s, may give insight into potential substrates that will allow us to follow phosphorylation events associated with Parkinson’s disease. In contrast to phosphorylation, a large number of proteins have been identified as being acetylated in mitochondria. We are interested in a broad range of acetylated proteins, but have listed [Pink1 and others] here based on either the large number of acetylated sites or the potential for acetylation to affect enzymatic activity. Structural determination of these proteins will provide insight into which of these sites may affect protein function so that we may focus our studies on these sites. Liza Pon Recessive missense mutations in the gene encoding for PINK1, a mitochondrial-localized serine/threonine kinase of unknown biological function, are a cause of familial Parkinson’s Disease. The role played by PINK1 is still elusive, but several studies have shown that mutant PINK1–/– Drosophila display enlarged and elongated mitochondria, a phenotype that can be rescued by the overexpression of parkin; this E3 ubiquitin ligase, when mutated, also causes a familial form of PD. In addition, recent studies have shown that PINK1 regulates mitochondrial dynamics by cooperating with other proteins, including parkin and miro (an adapter that links mitochondria to the microtubule-based motor kinesin). I would like to nominate PINK1 as a candidate for crystallization studies. PINK1 is likely to be a single pass membrane protein. The field would benefit from the structural information on both the soluble domains of PINK1 and of the membrane integral form of the protein. The kinase domain of PINK1 is expected to be similar to that of serine/threonine kinases. However, the C-terminal domain of this protein is known to regulate PINK1 kinase activity and is one of the sites of mutation in PD patients. Understanding the structure of the PINK1 C-terminal domain and its interactions with the kinase domain would allow us to develop models for regulation of PINK1 kinase activity by the C-terminal domain, and to develop a mutagenesis strategy to test those models. It will also reveal residues on the surface of PINK1, which may be important for interaction of PINK1 with parkin and miro. Finally, it will provide new insights in understanding how mutations that occur in PD affect the kinase activity, regulation and protein interactions of PINK1. cont’d next pg

MPP notes (dja): Function and Medical Relevance: Serine/threonine protein kinase, “Protects against mitochondrial dysfunction during cellular stress, potentially by phosphorylating mitochondrial proteins. Involved in the clearance of damaged mitochondria via selective autophagy (mitophagy). It is necessary for PARK2 recruitement to dysfunctional mitochondria to initiate their degradation.” (Uniprot) Physical Characteristics: Transit or Signal Peptides – transit peptide (1-77, Uniprot and TargetP; 1-28 DomainView). Functional Domains – protein kinase ( Uniprot, pfam), ATP-binding region ( ), ATP-binding site (186), proton-accepting active site (362). C3HC4-type RING finger binding domain inferred. TMs – predicted single pass ( Uniprot; HMMTop). PTMs - Predicted to autophosphorylate. Quaternary Structure – no information. Isoforms – this is canonical, isoform 2 is missing and are altered. Similar Structures: is 43% identical to 3e7oB (human MAP Kinase), is 43% identical to 2wntA (human ribosomal protein S6 kinase). Interactors: Substrates are ATP and unknown protein(s), products are ADP and phosphorylated protein(s), cofactors are Mg2+ and possibly Ca2+ (calcium-dependent protein kinase activity has been cited). Binds PARK7, PARK2, HTRA2, CDC37, HSP90AA1, ubiquitin, ubiquitin protein ligase/Parkin, Hsp75 mitochondrial precursor. Status: MPP – full-length was selected in MPP10 (Collaborator Workgroup #3) and is in cloning. NESG (ID HR6725) – large number of constructs generated that have not expressed, expressed insoluble. Other SG – none. Other: Human and mouse are 82% identical. Suggestions 1) Try in cell-free: -full-length - Nd77 (minus transit pep, uniprot and TargetP prediction) - Nd28 (minus transit pep, DomainView prediction) - C-term domain (e.g. 308-, corresponding to isoform 2) 2) Add mouse version?