1. MPP/TMPC OUTSIDE REQUEST FORM Written: 1/25/12 Requestor Reviewed: Requestor(s):Chuck HoppelExecs Reviewed: Institution:Case Western ReserveOutcome: Request Title: Human mitochondrial protein CPT1A, isoform 2 Protein NameORF IDSource OrganismHeatTarget Request IDTarget ScorepIResiduesSize (Da) CPT1AGO.118752Homo sapiens10TR.279628.775686239 Alt. names: Carnitine O-palmitoyltransferase 1, liver isoform | CPT1-L | Carnitine palmitoyltransferase 1A | CPT1 Requestor notes: The mitochondrial outer membrane and contact sites contain the enzyme carnitine palmitoyltransferase-1 (CPT-1) (a in liver and b in muscle tissues). This key enzyme is the control point for mitochondrial fatty acid oxidation. We have identified CPT-1 in a transfer complex in the outer membrane whereby antibodies directed towards specific peptides in CPT-1 result in the immunocapture of not only CPT-1 but also the long-chain activating enzyme and the voltage-dependent anion channel. The structure determination of CPT-1 is of great interest and we would be committed to doing function studies. MPP notes (dja): Function and Medical Relevance: See requestor notes. Reaction: Palmitoyl-CoA + L-carnitine = CoA + L-palmitoylcarnitine. Defects in CPT1A are the cause of carnitine palmitoyltransferase 1A deficiency (Uniprot). Physical Characteristics: Transit or Signal Peptides –none predicted. Functional Domains – carnityl-acyltransferase 170-763, CoA- and carnitine-binding sites, and proton acceptor/active site are mapped by similarity. TMs – 2, 48-73 and 103-122. PTMs - predicted by similarity; N-term Met removed, acetylation, phosphoryation. Quaternary Structure – see requestor notes, no other information. Isoforms – Isoforms 1 (canonical) differs from isoform 2 at the C- terminus (746-773: DSHRFGRHLKEAMTDIITLFGLSSNSKK → GIISQGPSSDT). See below, MPP is using isoform 2. Similar Structures: 34% identical over 169-767 to 2fy5A, human choline acetyltransferase. Binding Interactors: Proteins; ESR2, HSPA5, CARA, CLIC1, CYBA, NPDC1, GBAA, NR4A1, LEUD, KBTBD7. Small molecules: inhibitors malonyl-CoA, C75- CoA, CoA, more; substrates palmitoyl-CoA and carnitine; products CoA and palmitoylcarnitine. Literature: significant list in Uniprot including structure model. Activity Assay: presumably yes Status: CESG – worked extensively (outside request) with Nd165 form (GO.102091) in E. coli in vector pVP68k, 4 trials in 2008-2009, tiny amount purified; also mouse Nd165 (GO.102089, 87% identical) in E. coli in vector pVP68k; 4 trials 2008-2009, did not survive purification. MPP – chose isoform 2 as the clone was available. Cloned into pEU-cHis-GW and pEU-eXact, scored HWW and HWU respectively in standard cell-free, HHW with scaffolds. NESG – selected canonical isoform 1 (GO.111703, HR6758) and isoform 2 (GO.118752, HR8484), no further action. Other SG - none. Target Categories: Chosen: biomedical, membrane protein, PSI Biology partnership,structural coverage. Others: technology development, first structure of class, eukaryotic domain family, general domain family, protein family of high biological importance, disease, conformational state, functional mutant, complex with biological partner.

2. - Originally sent 1/25/12 - Forwarded again 2/28/12, suggesting conference call. Dear Dr. Hoppel, We do have somewhat encouraging results on CPT1A. In short, we cloned full-length isoform 2 into a wheat germ cell-free expression vector that adds a C-terminal His tag. As you probably know, isoform 2 has a slightly different C-terminus than isoform 1; we chose to work with it because this form was immediately available to us, but we would appreciate any advice you might have on the isoforms. In initial expression tests using our standard procedures, it expressed well but was only weakly soluble and purified poorly or not at all in small-scale IMAC purification tests.However, when coexpressed with "membrane scaffolding proteins", the protein became highly soluble, though it still purified poorly. These scaffolding proteins are likely forming lipid nanodiscs with lipids found in the wheat germ extract, and are hopefully incorporating CPT1A into the lipid disc.The poor purification may be the result of an inaccessible tag, which might be remedied by moving the tag to the N-terminus or, alternatively, using tags on the scaffolding proteins as a purification handle. In researching CPT1A I was surprised to find that our previous organization, the Center for Eukaryotic Structural Genomics, worked fairly extensively with N-terminal 165 residue-deleted versions of both human and mouse CPT1A. They were expressed in E. coli and we got as far as purifying several hundred micrograms which checked out by mass spectrometry (both mass and identity by ms/ms sequencing) but was not enough to attempt crystallization. At this point, we would like to obtain advice from you. It may be useful to take a look at the little write-up I've attached, which includes the description (in Requestor Notes) about CPT1A that you sent to Dave Pagliarini in your grant application support letter several years ago. Please correct any misconceptions I may have included and add anything that might be helpful to us, in particular any experience in expressing or purifying the protein or assaying activity. We would also be happy to teleconference with you if that would be useful. Tentative plans are to try some variations on the nanodisc production, possibly try production with liposomes and detergents, and potentially scale up production for which one goal would be to send you some protein for activity assay. We may also resurrect our E. coli effort since we now have some experience with membrane proteins that may produce a more successful outcome (note that the resources of MPP's sister center, the Transmembrane Protein Center, Brian Fox PI, will likely be brought to bear on this target as well). Regards, Dave Aceti