The determinants of chain type specificity and the mechanism of polyubiquitination by HECT E3s

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Title: The determinants of chain type specificity and the mechanism of polyubiquitination by HECT E3s
Author: Kim, Hyung Cheol
Abstract: Ubiquitination is a post -translational modification that can take several forms . Some proteins are modified with a single ubiquitin molecule , while others are modified with polyubiquitin chains . Each type of ubiquitination is thought to have distinct biological functions . The best -characterized types of ubiquitin modification are K48 -linked polyubiquitination , which serves as a signal for proteasomal degradation and K63 -linked polyubiquitination , which has non -proteolytic functions such in DNA repair , signaling , and endocytosis . HECT ubiquitin ligases (HECT E3s ) form a class of E3s , defined by a C terminal catalytic domain . Several lines of evidence suggested that the HECT E3s assemble a polyubiquitin chain in a sequential manner with one molecule of ubiquitin at a time being conjugated to the distal ubiquitin of the chain . In the process of chain elongation , not all HECT E3s target a common internal lysine of ubiquitin , leading to diversification of chain type specificity in HECT E3s . For example , yeast Rsp5 forms K63 chains , while human E6AP forms K48 chains . Two important mechanistic questions were addressed in my work : 1 ) what are the determinants of chain type specificity of HECT E3s , and 2 ) what allows the distal ubiquitin of a chain to be continuously oriented near the active site of the HECT domain in the course of a sequential polyubiquitination reaction ? I have determined that the chain type specificity of Rsp5 is a function solely of the HECT domain . Further , through the generation of chimeric HECT E3s , I demonstrated that chain type specificity determinants are located within the last 60 amino acids of the C lobe of the HECT domain . To address the second question , we solved the structure of Rsp5 HECT domain in complex with non -covalently bound ubiquitin in collaboration with Jue Chen’s laboratory (Purdue University ) . From the structure , we found that the N lobe of the HECT domain binds ubiquitin in a manner distinct from other known ubiquitin binding domains , and I have shown that Rsp5 proteins defective for ubiquitin binding are defective for chain elongation . We hypothesize that the ubiquitin binding site functions in the recruitment of the distal ubiquitin of polyubiquitin chain for efficient polyubiquitination .
URI: http : / /hdl .handle .net /2152 /ETD -UT -2010 -12 -2427
Date: 2011-01-26

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The determinants of chain type specificity and the mechanism of polyubiquitination by HECT E3s. Doctoral dissertation, University of Texas at Austin. Available electronically from http : / /hdl .handle .net /2152 /ETD -UT -2010 -12 -2427 .

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