A Human Ubiquitin Conjugating Enzyme (E2)-HECT E3 Ligase Structure-function Screen

Here we describe a systematic structure-function analysis of the human ubiquitin (Ub) E2 conjugating proteins, consisting of the determination of 15 new high-resolution three-dimensional structures of E2 catalytic domains, and autoubiquitylation assays for 26 Ub-loading E2s screened against a panel of nine different HECT (homologous to E6-AP carboxyl terminus) E3 ligase domains. Integration of our structural and biochemical data revealed several E2 surface properties associated with Ub chain building activity; (1) net positive or neutral E2 charge, (2) an “acidic trough” located near the catalytic Cys, surrounded by an extensive basic region, and (3) similarity to the previously described HECT binding signature in UBE2L3 (UbcH7). Mass spectrometry was used to characterize the autoubiquitylation products of a number of functional E2-HECT pairs, and demonstrated that HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. Our data set represents the first comprehensive analysis of E2-HECT E3 interactions, and thus provides a framework for better understanding the molecular mechanisms of ubiquitylation. Here we describe a systematic structure-function analysis of the human ubiquitin (Ub) E2 conjugating proteins, consisting of the determination of 15 new high-resolution three-dimensional structures of E2 catalytic domains, and autoubiquitylation assays for 26 Ub-loading E2s screened against a panel of nine different HECT (homologous to E6-AP carboxyl terminus) E3 ligase domains. Integration of our structural and biochemical data revealed several E2 surface properties associated with Ub chain building activity; (1) net positive or neutral E2 charge, (2) an “acidic trough” located near the catalytic Cys, surrounded by an extensive basic region, and (3) similarity to the previously described HECT binding signature in UBE2L3 (UbcH7). Mass spectrometry was used to characterize the autoubiquitylation products of a number of functional E2-HECT pairs, and demonstrated that HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. Our data set represents the first comprehensive analysis of E2-HECT E3 interactions, and thus provides a framework for better understanding the molecular mechanisms of ubiquitylation. Ubiquitin (Ub) 1The abbreviations used are:UbubiquitinUBCubiquitin conjugatingHECThomologous to E6-AP carboxyl terminus. 1The abbreviations used are:UbubiquitinUBCubiquitin conjugatingHECThomologous to E6-AP carboxyl terminus. is a conserved polypeptide that is covalently conjugated to other proteins in a reversible manner, to alter their function in a variety of ways. Ub conjugation (ubiquitylation) is a highly regulated process, consisting of a sequential series of E1-E2-E3 activation, conjugation, and ligation reactions. An E1 enzyme must first activate a mature Ub polypeptide via the formation of a high-energy thiol-ester bond with the Ub carboxyl-terminal Gly residue. The activated Ub polypeptide is then transferred to a Cys residue of an E2 conjugating protein. Finally, via an E3 ligase, the Ub polypeptide is covalently conjugated to a target protein (reviewed in 1Schwartz A.L. Ciechanover A. Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology.Annu. Rev. Pharmacol. Toxicol. 2009; 49: 73-96Crossref PubMed Scopus (361) Google Scholar, 2Deshaies R.J. Joazeiro C.A. RING domain E3 ubiquitin ligases.Annu. Rev. Biochem. 2009; 78: 399-434Crossref PubMed Scopus (1875) Google Scholar, 3Finley D. Recognition and processing of ubiquitin-protein conjugates by the proteasome.Annu. Rev. Biochem. 2009; 78: 477-513Crossref PubMed Scopus (1275) Google Scholar, 4Pickart C.M. Eddins M.J. Ubiquitin: structures, functions, mechanisms.Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (1021) Google Scholar, 5Pickart C.M. Fushman D. Polyubiquitin chains: polymeric protein signals.Curr. Opin. Chem. Biol. 2004; 8: 610-616Crossref PubMed Scopus (830) Google Scholar). ubiquitin ubiquitin conjugating homologous to E6-AP carboxyl terminus. ubiquitin ubiquitin conjugating homologous to E6-AP carboxyl terminus. Monoubiquitylation (i.e. the conjugation of a single Ub molecule to a target protein) has been implicated in a number of biological processes including transcriptional control, endocytosis, plasma membrane receptor recycling, and DNA damage signaling (6Saksena S. Sun J. Chu T. Emr S.D. ESCRTing proteins in the endocytic pathway.Trends Biochem. Sci. 2007; 32: 561-573Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 7Mosesson Y. Yarden Y. Monoubiquitylation: a recurrent theme in membrane protein transport.Isr. Med. Assoc. J. 2006; 8: 233-237PubMed Google Scholar). However, Ub itself contains seven lysine residues, all of which can be ubiquitylated to form polyubiquitin oligomers, or Ub “chains” (2Deshaies R.J. Joazeiro C.A. RING domain E3 ubiquitin ligases.Annu. Rev. Biochem. 2009; 78: 399-434Crossref PubMed Scopus (1875) Google Scholar, 3Finley D. Recognition and processing of ubiquitin-protein conjugates by the proteasome.Annu. Rev. Biochem. 2009; 78: 477-513Crossref PubMed Scopus (1275) Google Scholar, 4Pickart C.M. Eddins M.J. Ubiquitin: structures, functions, mechanisms.Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (1021) Google Scholar, 5Pickart C.M. Fushman D. Polyubiquitin chains: polymeric protein signals.Curr. Opin. Chem. Biol. 2004; 8: 610-616Crossref PubMed Scopus (830) Google Scholar, 8Ye Y. Rape M. Building ubiquitin chains: E2 enzymes at work.Nat. Rev. Mol. Cell Biol. 2009; 10: 755-764Crossref PubMed Scopus (692) Google Scholar). Ub chains of varying lengths and linkage types can confer very different biological outcomes to a targeted protein substrate. For example, the best-characterized function of Ub chains (in this case, consisting of at least four Ub polypeptides linked via K48) is the targeting of a protein substrate for 26S proteasome-dependent degradation (9Thrower J.S. Hoffman L. Rechsteiner M. Pickart C.M. Recognition of the polyubiquitin proteolytic signal.EMBO J. 2000; 19: 94-102Crossref PubMed Scopus (1309) Google Scholar). By contrast, K63-linked Ub chains play roles in the DNA damage response, epsin-mediated endocytosis and aggresome formation (10Acconcia F. Sigismund S. Polo S. Ubiquitin in trafficking: the network at work.Exp. Cell Res. 2009; 315: 1610-1618Crossref PubMed Scopus (163) Google Scholar, 11Chin L.S. Olzmann J.A. Li L. Parkin-mediated ubiquitin signalling in aggresome formation and autophagy.Biochem. Soc Trans. 2010; 38: 144-149Crossref PubMed Scopus (106) Google Scholar). Although only two human Ub E1 activating proteins have been identified (12Jin J. Li X. Gygi S.P. Harper J.W. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging.Nature. 2007; 447: 1135-1138Crossref PubMed Scopus (263) Google Scholar), 40 E2s (including both active E2 proteins and inactive E2 variants) are encoded in the human genome (Table I). All E2 proteins share a conserved “core” ubiquitin conjugating (UBC) domain of ∼150 amino acid residues, and many E2s possess additional N- and/or C-terminal protein sequences that can govern intracellular localization, confer regulatory properties, or provide specificity for interactions with particular E3 ligases (13Wenzel D.M. Stoll K.E. Klevit R.E. E2s: structurally economical and functionally replete.Biochem. J. 2011; 433: 31-42Crossref PubMed Scopus (89) Google Scholar, 14Pickart C.M. Mechanisms underlying ubiquitination.Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2909) Google Scholar).Table IE2 (including both active and inactive E2 variants) and HECT domain protein information. Indicated are gene name, aliases, protein length, PDBID code (if structure has been solved) and NCBI protein and gene accession numbers. “Protein Note” we to or protein. the of E2 protein to form a bond with in our in a new Ub E3 ligases the of Ub from an activated E2 to a substrate protein or Ub of the RING new E3 activated to in Ub from the E2 to the target (2Deshaies R.J. Joazeiro C.A. RING domain E3 ubiquitin ligases.Annu. Rev. Biochem. 2009; 78: 399-434Crossref PubMed Scopus (1875) Google Scholar), of the HECT (homologous to E6-AP carboxyl terminus) domain E3 form a thiol-ester linkage with Ub to to a target protein F. M. Ciechanover A. The HECT of E3 ubiquitin in Full Text Full Text PDF PubMed Scopus Google Scholar, D. S. of the HECT of ubiquitin Rev. Mol. Cell Biol. 2009; 10: PubMed Scopus Google Scholar). The human genome of RING only HECT E3 ligases D. S. of the HECT of ubiquitin Rev. Mol. Cell Biol. 2009; 10: PubMed Scopus Google Scholar). specificity for of E2 proteins, and different can different types of Ub chains K.E. S. J. Rape M. The of ubiquitin chain by a 2011; Full Text Full Text PDF PubMed Scopus Google Scholar, L. A. S. Rape M. of formation by the human Full Text Full Text PDF PubMed Scopus Google Scholar, Klevit R.E. RING interactions of or polyubiquitin chain Mol. Biol. 2007; PubMed Scopus Google Scholar, A. M. M. The activation of the E3 ligase function of the of J. PubMed Scopus Google Scholar). However, the molecular in E2-HECT E3 specificity are the properties associated with (i.e. the of Ub are and the molecular mechanisms in the of different types of Ub of the of interactions, have been largely in the of functional Although two has identified a number of functional interactions S. A. J. J. S. M. D. L. C.M. of the human E2 ubiquitin conjugating enzyme protein Res. 2009; 19: PubMed Scopus Google Scholar, A. comprehensive framework of E3 interactions of the human Biol. 2009; PubMed Scopus Google Scholar), this provide or Ub chain linkage types by with proteins must be used to this of information. interactions a number of RING domain and a number of E2 proteins have been previously K.E. S. J. Rape M. The of ubiquitin chain by a 2011; Full Text Full Text PDF PubMed Scopus Google Scholar, L. A. S. Rape M. of formation by the human Full Text Full Text PDF PubMed Scopus Google Scholar, Klevit R.E. RING interactions of or polyubiquitin chain Mol. Biol. 2007; PubMed Scopus Google Scholar, A. M. M. The activation of the E3 ligase function of the of J. PubMed Scopus Google Scholar), has the HECT E3 we the first comprehensive human E2-HECT E3 structure function E2 by from in and the a with and a or located of the HECT domain proteins (Table and Ub The of E2 be and/or other in in in the of at to an of with and for at 15 E2 and HECT domain proteins with to All proteins for the Ub assays against and at For of the E2 proteins, the was by with at by a the target protein and by an with to a protein of E2 protein at the in with to and in data an or at at the or the with M. of data in PubMed Scopus Google Scholar), the Biol. 2010; PubMed Scopus Google Scholar), or J.W. The in data Biol. PubMed Scopus Google Scholar). All structures, other that of and by molecular J. of molecular in Biol. 2001; PubMed Scopus Google Scholar), R.J. 2007; PubMed Scopus Google or A. A. with Biol. 2010; PubMed Scopus Google Scholar). The structure of and by single the or J. and structure Biol. PubMed Scopus Google Scholar). building was R.J. A. and of protein PubMed Scopus Google or Biol. 2000; PubMed Scopus Google Scholar). building was the for molecular Biol. 2004; PubMed Scopus Google with with of structures by the Biol. PubMed Scopus Google Scholar), J.S. J. M. R.J. T. system: new for structure Biol. PubMed Scopus Google Scholar), or R.J. J.S. a comprehensive for structure Biol. 2010; PubMed Scopus Google Scholar). was of the for protein D. PubMed Scopus Google Scholar). All structures have been and can be in E2 assays in a of of and of Ub in a consisting of 40 and for at by the of by and by a against the an and (in the of in a of in a of and E1 E2 ubiquitin and E3 HECT domain proteins, at for by the of and the of and with E2 of or of and at for proteins by against of a of human E2 conjugating enzymes the of The of the structures of UBE2L3 and used for surface analysis J. The Res. 2000; PubMed Scopus Google Scholar). An was used for All of the in the only is for in the structural for human E2 conjugating enzymes that an was to seven and domain and and other and that the E2 for by at a of and Li and of structures for molecular Res. 2007; PubMed Scopus Google with the D. of Chem. Scopus Google the represents and the is the is the is the is the is the charge, is the is the and is the or located at J.A. in structure and Rev. Scopus Google Scholar). The by E2 enzyme in a and the to at charge, and at that D. S. M.J. J.A. of to and the Sci. S. A. 2001; PubMed Scopus Google Scholar). The surface was a with a of and surface was a with a of a all of the E2 enzymes and to enzyme the at the molecular D.M. for and Chem. 2004; PubMed Scopus Google Scholar). of the of of the E2 enzymes by a similarity to the and are of proteins and at a and the number of that proteins a of have of a of have different of the similarity was with a and linkage the An of Biol. PubMed Scopus Google Scholar, of 2004; 32: Scopus Google Scholar), which and and Our analysis was The by (in (in and a by data (i.e. with a The represents the that a particular in the and from the other E2 which are two and two to a net and and a net positive and to the and of for the domains, additional of the and two for and to with for and the proteins at was in M. S. M. L. J. J. or a Biol. 10: PubMed Scopus Google for The was to an to a products from from and with first a and was a from with to with at a of The was in was and the for in the for which two been for to the with M. S. of spectrometry data and to 2004; PubMed Scopus Google Scholar), and data proteins with 2004; PubMed Scopus Google Scholar, the for protein Mol. Biol. 2006; Google against the and against our previously T. ubiquitin and protein 2010; 10: PubMed Scopus Google Scholar), with additional from and for a of and an of with to two for of lysine was a to the of and of of was used a to a of J.S. and of a for from 2007; PubMed Scopus Google Scholar, J.S. Building for in PubMed Scopus Google was used for with a of used a to a of have all to analysis of in autoubiquitylation assays to with the associated for in Ub linkage was used to and of of all seven polypeptides the and the with a and of of or and of the with a target of was a of and the for Ub linkage was all was then the of the for linkage with to the linkage with the An was from the and this to all data the of a conserved catalytic domain of ∼150 we a the human E2 proteins this E2s the and the E2s that the conjugation of proteins are from that Ub conjugation the very in For example, the E2 for the by Rev. Biochem. 2004; PubMed Scopus Google Scholar, F. Rev. Cell Biol. 2000; PubMed Scopus Google Scholar), is with two Ub and and E2s for the L. of the activating and conjugating enzymes of the conjugation Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), are the a Ub E2 of binding and Mol. Biol. 2007; PubMed Scopus Google Scholar). similarity and thus to have in E2 functional better the human we a E2 protein and “core” of the 40 E2 proteins a (Table I). both and polypeptides for different E2s in and to The proteins or we to the domains of of the E2s (Table I). E2 proteins of the 40 domains in the human form the of our the of human E2 enzymes to Although the structures of a number of E2s have been (Table from several of the of the E2 have been previously better E2 structure-function we high-resolution three-dimensional structures of 15 additional human E2 domains (Table and structures the number of E2 domains, with that have been previously provide structural of the human E2 of the new structures a E2 of a four surrounded by The domains of E2s share a of similarity in three-dimensional with an of of to the of the protein E2s and are that the polypeptide structure for conjugating surface properties or additional structural must be for conserved in the (1) the catalytic the catalytic Cys in the (2) the E3 binding region, and the and and (3) the of and in all E2 conserved of the the the E3 ligase binding and the catalytic and and Our E2 panel was to an in to the of conjugating enzyme to form a the of and the E1 enzyme we that 26 of the human E2s of Ub is in with with was to Ub in our Our Ub revealed that and the of an E3 protein to Ub conjugation (i.e. the formation of an bond with the of a lysine residue in of the proteins in the our in we Ub for the protein E2s and the inactive Ub E2 which a catalytic or in our with a that this E2 is by an Ub (12Jin J. Li X. Gygi S.P. Harper J.W. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging.Nature. 2007; 447: 1135-1138Crossref PubMed Scopus (263) Google interactions the set of Ub E2s and a number of HECT E3 ligases HECT domains (Table including of the and the and and other HECT domains and proteins in to and in an autoubiquitylation with of the 26 Ub-loading E2 at via and ubiquitylated products by and Ub in we of the autoubiquitylation of (1) Ub chains (2) Ub or and (3) with products and functional interactions with all of the HECT domains, with and of interactions highly in the of Ub was to catalyze the of chains with and and chains or with several other and functionally with several of the HECT domains to chains or and only of in our and interactions with for the first a human E2-HECT functional a we in the and of an For example, the domains of and with their in autoubiquitylation reactions. the protein was active in the data that additional sequences the can positive or regulatory domains and/or provide additional E3 that confer Ub chain building and functional interactions with HECT domains, surface properties of E2 structure and for E2 and the was used to at for the of the highly active E2s identified in our HECT autoubiquitylation an net positive or neutral charge, the E2s a net revealed that of the highly active E2s possess a “acidic trough” to the catalytic residue to and in located in the and surrounded by extensive basic of the of the active the of of the ubiquitin to and in the a of The active E2s in thus share a of similarity the Ub binding with a net charge, and properties from other E2 By contrast, the surface of the E3 binding and Ub of in our have that E2 to and the in to by the J. D.M. D. ubiquitin from a structure of a 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). to the catalytic to the binding of the in the the of Ub M.J. S. M. of a conjugating a for the ubiquitin 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the a in we a series of proteins in which amino in this to basic a of E2 the of Ub a autoubiquitylation with the HECT domain was of the Ub in the conjugates to with of for interactions and Ub conjugation and of to basic amino and for residue in in this and with other HECT domains data are with an of the the catalytic Cys was for Although E2s can catalyze with RING E3 UBE2L3 is by HECT of binding and Mol. Biol. 2007; PubMed Scopus Google Scholar, D.M. A. Klevit R.E. and to be 2011; PubMed Scopus Google Scholar). The HECT domain protein E6-AP with UBE2L3 and with a of and to other E2s with of binding and Mol. Biol. 2007; PubMed Scopus Google Scholar). The UBE2L3 for binding to the E6-AP HECT domain previously identified and this all of the active Ub E2s in our and for HECT binding the binding associated with a number of UBE2L3 of binding and Mol. Biol. 2007; PubMed Scopus Google and the active E2s in our HECT in this that proteins possess structural for HECT domain binding other human E2 of structural similarity to the E6-AP binding domain of UBE2L3 was thus an of Ub chain building in in with HECT domains. this signature is for E2 in autoubiquitylation we a series of UBE2L3 proteins in which in the E6-AP HECT domain and their in with the E3 ligase HECT domain UBE2L3 proteins at in the E6-AP binding and for residue a in with data that the of the E2 protein for HECT domains an in ubiquitylation. Finally, we spectrometry to characterize the Ub chain by a number of functional products via and proteins at to The spectrometry identified two (1) with the of a to the Ub a Gygi S.P. in the of Biol. PubMed Scopus Google Scholar), and (2) a that we and which contains from all seven Ub chain linkage types T. spectrometry to ubiquitin and protein conjugation 2009; PubMed Scopus Google Scholar, T. ubiquitin and protein 2010; 10: PubMed Scopus Google Scholar). or with the RING or a of Ub was in which all other linkage types and UBE2L3 was functional with of RING the of the the HECT domains from and the formation of a of and Ub of was all other linkage types and contrast, the HECT domain to a very different of Ub chains with the set of Ub that in all other very different linkage types in the different HECT the of HECT domains in Ub linkage and both of which have been previously demonstrated to Ub chains in R.J. of lysine by the Full Text Full Text PDF PubMed Scopus Google Scholar, Pickart C.M. and function of ubiquitin conjugating enzyme the is a PubMed Scopus Google Scholar), in (1) (2) the RING or and (3) HECT domains from and of E2s the of Ub highly in in the of or with RING and linkage was in However, or with HECT was inactive with the of Ub in the to with and chain with of and with a of linked Ub and with by HECT and the of chain Biol. 2009; PubMed Scopus Google Scholar, M. Pickart C.M. HECT domain ubiquitin ligases mechanisms of polyubiquitin chain J. PubMed Scopus Google Scholar), our data thus (1) HECT domains can govern linkage in autoubiquitylation largely independent of E2 linkage and (2) different HECT domains different linkage we that the of Ub in the HECT domains and Ub chains consisting of and of the human E2 that the is a of and an “acidic trough” near the catalytic surrounded by extensive basic surface is in a number of roles in The of the near the catalytic Cys with and the amino to the activation of the substrate lysine activation and functional analysis of conjugation in the Mol. Biol. 2006; PubMed Scopus Google Scholar). demonstrated that of two in this and of the of from to a target lysine activation and functional analysis of conjugation in the Mol. Biol. 2006; PubMed Scopus Google Scholar). The of the from the be in and/or the polypeptide in both the and the structures, this of the E2 with the M.J. S. M. of a conjugating a for the ubiquitin 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, activation and functional analysis of conjugation in the Mol. Biol. 2006; PubMed Scopus Google Scholar). with this we that of in the in HECT reactions. to all For example, was highly active in our autoubiquitylation very different surface structural confer a function in this have that a in an in the of polyubiquitin chains R.J. of lysine by the Full Text Full Text PDF PubMed Scopus Google Scholar). that similarity to the previously HECT binding of UBE2L3 was a of for all E2s in in and of binding and Mol. Biol. 2007; PubMed Scopus Google first the of UBE2L3 in binding to the HECT E6-AP and the J. D.M. D. ubiquitin from a structure of a 2009; Full Text Full Text PDF PubMed Scopus Google structure the that the E2s the similarity with UBE2L3 in this of Ub chain building in autoubiquitylation with a number of different HECT domains. analysis of the HECT binding signature in UBE2L3 that this an in Ub with all HECT and E6-AP the E2 protein via and J. D.M. D. ubiquitin from a structure of a 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). However, E6-AP to extensive interactions with UBE2L3 via the and E6-AP and possess E2 binding and in this the in chain linkage types by the with the E2 proteins, the binding to E2s the in a that the formation of Ub with this autoubiquitylation interactions with the set of E2s and Ub linkage in that the E2 for be be to this have that HECT linkage specificity in autoubiquitylation by HECT and the of chain Biol. 2009; PubMed Scopus Google Scholar, M. Pickart C.M. HECT domain ubiquitin ligases mechanisms of polyubiquitin chain J. PubMed Scopus Google Scholar), possess a catalytic Cys residue. However, many different can be For example, (1) a single Ub molecule is transferred to the HECT Cys Ub chains be by this case, the E2 the linkage (2) Ub chains by or E2s be transferred to a HECT conjugation to a specificity be by the E2 in this (3) an E2 with a HECT is that chain types by the other that we are we several different HECT domains with two E2s that Ub chains in and Klevit R.E. RING interactions of or polyubiquitin chain Mol. Biol. 2007; PubMed Scopus Google Scholar, M. Pickart C.M. HECT domain ubiquitin ligases mechanisms of polyubiquitin chain J. PubMed Scopus Google that the chain types in autoubiquitylation different in in an with that the HECT domain is the of in this from the Rape that in with of Ub itself to Ub chain K.E. S. J. Rape M. The of ubiquitin chain by a 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). the Ub and is in with the active and of chains in and in or with other E2s function in a manner, we all E2 structures, and in this other E2s possess at the that with Ub in our understanding that specificity least a is a only to Finally, is to that functional E2-HECT that can only Ub chains or that or be in our our new set of E2 structures, with the of a E2 protein a comprehensive E2-HECT functional and a Ub linkage data represents an for better understanding the structural properties that E2-HECT interactions, and the of different types of Ub chain F. and Y. Li for with A. for of the in this are from at and at the is by for the of of and has been in or in with from the and the of of the was by the of the a with data was at is by the via and the is by with

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