Multiple intermediates on the energy landscape of a 15-HEAT-repeat protein

• Published in: Structure (London) 2013-10, Vol.21 (11), p.1954-1965
• Main Authors: Tsytlonok, Maksym, Craig, Patricio O., Sivertsson, Elin, Serquera, David, Perrett, Sarah, Best, Robert B., Wolynes, Peter G., Itzhaki, Laura S.
• Format: Article
• Language: English
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2013.08.028

Repeat proteins are a special class of modular, non-globular proteins composed of small structural motifs arrayed to form elongated architectures and stabilised solely by short-range contacts. We find a remarkable complexity in the unfolding of the large HEAT repeat protein PR65/A. In contrast to what has been seen for small repeat proteins in which unfolding propagates from one end, the HEAT array of PR65/A ruptures at multiple distant sites, leading to intermediate states with non-contiguous folded subdomains. Kinetic analysis allows us to define a network of intermediates and to delineate the pathways that connect them. There is a dominant sequence of unfolding, reflecting a non-uniform distribution of stability across the repeat array; however the unfolding of certain intermediates is competitive, leading to parallel pathways. Theoretical models accounting for the heterogeneous contact density in the folded structure are able to rationalize the variation in stability across the array. This variation in stability also suggests how folding may direct function in a large repeat protein: The stability distribution enables certain regions to present rigid motifs for molecular recognition while affording others flexibility to broaden the search area as in a fly-casting mechanism. Thus PR65/A uses the two ends of the repeat array to bind diverse partners and thereby coordinate the dephosphorylation of many different substrates and of multiple sites within hyperphosphorylated substrates.