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Structure, Function and Inhibition of the Prohormone/Proprotein Convertase Family of Endoproteinases

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http://pubman.mpdl.mpg.de/cone/persons/resource/persons78791

Than,  Manuel E.
Huber, Robert / Structure Research, Max Planck Institute of Biochemistry, Max Planck Society;

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Than, M. E. (2006). Structure, Function and Inhibition of the Prohormone/Proprotein Convertase Family of Endoproteinases. Habilitation Thesis, Ludwig-Maximilians-Universität, München.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0010-64D9-3
Abstract
In eukaryotes, many secreted proteins and peptides are proteolytically excised from larger precursors by calcium dependent serine proteinases, the proprotein / prohormone convertases (PCs). This cleavage is essential for the activation of the respective substrates, ranging from peptide hormones (such as insulin), extracellular proteinases, growth and differentiation factors (implicated in neurodegenerative diseases, tumor growth and metastasis) to bacterial toxins and viral coat proteins. Hence, this class of endoproteinases represents a very interesting pharmacological target. The seven mammalian PCs and their yeast orthologue kexin are multi domain proteinases that cleave their protein substrates very specifically after multiple basic residues. They consist of a subtilisin related catalytic domain, a conserved P-domain and a variable, often cysteine rich domain, which is followed by a transmembrane helix and a short cytoplasmic domain in some PCs. Furin, the best studied mammalian family member, cleaves its substrates after basic Arg-Xaa-Arg/Lys-Arg like motifs. We have crystallized and solved the 2.6 Å crystal structure of the decanoyl-Arg-Val-Lys-Argchloromethylketone (dec-RVKR-cmk) inhibited mouse furin ectodomain, revealing an eightstranded jelly-roll P-domain tighly associated with the catalytic domain. The active site cleft is an extended, narrow but deep depression presenting highly specific interaction subsites for substrate recognition. A large number of negative surface charges is concentrated within this cleft, especially within the tight binding S1 pocket. Based on modeling studies, we have extended this study to the entire PC-family, showing a highly similar overall structure of all family membres, but differences in the more distant specificity subsites and in the number and distribution of negative charges within the active site cleft. Special focus was given to PC2, which shows a completely different northern ridge of the S4 pocket. The autoproteolytic maturation of furin has been studied by modeling techniques, resulting in a comprehensive structural model for pro-furin and its stepwise activation. This activation process might also be applicable to other family members, except for PC2, which is activated by an even more complicated series of events. Two calcium binding sites have been unequivocally identified in furin by using a novel type of double difference Fourier map, that leads directly to an element specific electron density. While Ca-1 fulfills a well conserved structural function also in many bacterial digestive subtilisins, Ca-2 is unique to the seven known PC family members and is specifically required to stabilize the high number of negative charges at the S1 pocket. The development of PC-specific inhibitors of potential anti-bacterial and anti-viral activity by rational structure based methods has been initiated. In a second study, we have analyzed the forces within the dimeric interface between the non-collagenous (NC1) domains of the tight type IV collagen network, which stabilizes the sheet-like basement membranes and presents anchoring sites for various other membrane constituents. Our crystal structure of isolated NC1 particles shows that two trimeric cap-like structures interact via a large interface, stabilizing this dimeric contact. Each cap represents an internally completed and highly interdigitated novel fold, ruling out the existence of domain swapping or difulfide exchange reactions across this interface as suggested previously to occur upon dimer formation. Based on extensive biochemical and crystallographic investigations we describe the presence of a novel type of covalent cross link between the side chains of directly opposing methionine and lysine side chains, that further stabilizes this dimeric interface and should be responsible for the biochemically observed "chain dimers" that originally gave rise to the disulfide exchange model. The structures of two additional proteinases have been investigated, the 'digestive' KDEL-tailed cysteine proteinase of Ricinus communis endosperm, which plays a central role in plant tissue senescence and the mast cell derived tetrameric trypsin-like serine proteinase tryptase, which shows an interesting structural transition between the inactive α- and the active β-form.