An NMR-based Model of the Ubiquitin-bound Human Ubiquitin ...

13154The Structural Basis for Lysine 63 Chain CatalysisFIG. 3. Binding-induced NMR chemical shift perturbationanalysis of hUbc13 with Ub. Comparison of backbone amide 1 H and15 N chemical shift of hUbc13 in the absence or presence of thiolesterlinkedUb (A) and hMms2 (B) or the comparison between hMms2 15 N-hUbc13 heterodimer and this heterodimer in the presence of thiolesterlinkedUb (C). The total change in chemical shift, total , was calculatedfor hUbc13 under each of the conditions and plotted as a function ofprimary amino acid sequence. Dashed lines represent the averagechange in total as well as one standard deviation above this average.Residues whose change in chemical shift could not be identified areindicated with an asterisk.which provide a measure of the deviation between the observedchemical shifts and their random coil values, and are indicativeof the type of secondary structure (37, 38). A comparison betweensecondary structural elements for hMms2 and hUbc13,determined by x-ray crystallography to those determined fromthe chemical shift indices, demonstrate a close correlation betweentypes of secondary structure determined in the solutionand crystalline states.The Non-covalent Interaction between hMms2 and Ub—BothhMms2 and hUbc13 have each been observed to exist in amonomeric state and as the heterodimer (23, 27), whereashomodimerization has not been observed (see “ExperimentalProcedures”), and therefore an examination of the interactionbetween Ub and the hMms2 subunit is of interest. The chemicalshift perturbations that result from the interaction of 15 N-hMms2 subunit with unlabeled acceptor Ub are shown in Fig.2A. The greatest effects on total upon interaction with Ub areobserved at the N-terminal portion of hMms2. Specifically, theaffected residues are located in helix 1 (Glu-20, Gly-22, Lys-24), sections of strand 1 (Val-31, Ser-32, Leu-35), strand 2(Thr-47, Gly-48, Met-49), strand 3 (Tyr-63, Leu-65), helix 2(Leu-119) as well as the loop joining helix 1 to strand 1(Val-26, Thr-30). Intermediate effects on total are found closein sequence to the greatest changes and include the C-terminalportion of 1 (Gln-23), sections of 1 (Trp-33), 2 (Trp-46), L2prior to 3 (Asn-60, Arg-61), 3 (Val-67, Gly-70), and the loopjoining 1 to1 (Gly-25, Gly-27, Gly-29). Intermediate changesare also found in 2 (Gln-120, Leu-125, Glu-130) and the Cterminus (Gly-140, Gln-141).As expected, many of the residues in hMms2 that exhibit thegreatest backbone amide chemical shift perturbations are locatedon the surface of the protein, and contain surface exposedside chains that may be involved in non-covalent interactionswith Ub (Fig. 5A). These residues cluster onto one face ofhMms2, forming three distinct patches. Interestingly, no significantchanges in chemical shift were observed for residues onthe opposite surface of hMms2. The first patch is perpendicularto the hUbc13hMms2 interface, and is composed of residues atthe C-terminal end of 1 and the loop that joins 1 to 1(Glu-20, Glu-21, Gly-22, Gln-23, Lys-24, Gly-25, Val-26, Gly-27,Gly-29, and Val-31), portions of 1 (Ser-32, Trp-33, and Leu-35), 2 (Thr-47, Gly-48, and Met-49), and 3 (Arg-61, Tyr-63,and Leu-65). The second patch is found at the C-terminalportion of hMms2. Notably, the total surface area of boththese hMms2 patches corresponds well with the complementarypatch on Ub that has previously been demonstrated tointeract with hMms2 (27). Additionally, the combined electrostaticsurface potential of the hMms2 patches is complementaryto that found on Ub (Fig. 5C). Interestingly, thethird patch involves hMms2 residues that would normallyinteract with hUbc13 in the heterodimer, and include Val-7,Lys-8 (greatest total ), and other N-terminal amino acids ofhMms2 (intermediate total ).Our previous findings indicated that the Ub contact surfacewith hMms2 remained largely the same when alone or incomplex with hUbc13 (27). When we next examined the 15 N-hMms2-Ub interaction as a heterodimer with hUbc13 we similarlyfound that the hMms2 residues that undergo change onUb binding closely parallel those of the individual subunit withsome notable exceptions (Fig. 2C). As with hMms2 alone, manyof the major total are found near the C terminus of 1 (Glu-20,Gln-23), the loop that joins it to 1 (Val-26, Gly-29, Thr-30), 1(Val-31), 2 (Gly-48, Met-49), and 3 (Arg-61, Tyr-63, Leu-65).Residues with intermediate values of total are also similar,including 1 (Leu-19, Glu-21), the loop joining 1 to1 (Gly-25), 1 (Trp-33, Gly-34), 2 (Thr-47, Gly-52), 3 (Asn-60, Ile-62,Val-67), 2 (Ser-114, Ile-115, Val-117, Gln-120, Leu-125, Glu-130), and the C terminus (Gln-141). The backbone amide 1 H N -15 N HSQC NMR cross-peaks for three residues (L1 (Asp-37)and 2 (Arg-45, Ile-50)) either experienced large changes inchemical shift, rendering identification difficult, or their intensitieswere severely diminished due to line-broadening as aresult of complex formation.In contrast to the hMms2 subunit alone, none of the N-terminal residues situated at the heterodimer interface undergosignificant change upon Ub binding, whereas significantchange is detected within L1 (Asp-38, Asp-40, Met-41, andArg-45). Notably, the region surrounding the vestigial activesite of hMms2 does not appear to play a role in Ub binding. Thisresult clearly distinguishes the hMms2-Ub interaction fromother previously reported E2-Ub interactions. The changes inthe surface characteristics of the hMms2 component of theheterodimer upon Ub binding are shown in Fig. 5B.Downloaded from www.jbc.org at University of British Columbia on February 18, 2009