An NMR-based Model of the Ubiquitin-bound Human Ubiquitin Conjugation Complex Mms2·Ubc13

THE STRUCTURAL BASIS FOR LYSINE 63 CHAIN CATALYSIS*,

Sean McKennaDagger §, Trevor MoraesDagger , Landon Pastushok||, Christopher PtakDagger , Wei Xiao||**, Leo SpyracopoulosDagger Dagger Dagger §§, and Michael J. EllisonDagger Dagger Dagger ¶¶

From the Dagger  Department of Biochemistry and the Dagger Dagger  Institute for Biomolecular Design, University of Alberta, Edmonton, Alberta T6G 2H7 and the || Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada

Received for publication, December 4, 2002, and in revised form, February 4, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A heterodimer composed of the catalytically active ubiquitin-conjugating enzyme hUbc13 and its catalytically inactive paralogue, hMms2, forms the catalytic core for the synthesis of an alternative type of multiubiquitin chain where ubiquitin molecules are tandemly linked to one another through a Lys-63 isopeptide bond. This type of linkage, as opposed to the more typical Lys-48-linked chains, serves as a non-proteolytic marker of protein targets involved in error-free post-replicative DNA repair and NF-kappa B signal transduction. Using a two-dimensional 1H-15N NMR approach, we have mapped: 1) the interaction between the subunits of the human Ubc13·Mms2 heterodimer and 2) the interactions between each of the subunits or heterodimer with a non-covalently bound acceptor ubiquitin or a thiolester-linked donor ubiquitin. Using these NMR-derived constraints and an unbiased docking approach, we have assembled the four components of this catalytic complex into a three-dimensional model that agrees well with its catalytic function.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The post-translational modification of intracellular proteins by ubiquitination fulfills an important regulatory function in many cellular pathways. Protein ubiquitination involves a cascade of enzymatic steps where ubiquitin (Ub)1 is passed sequentially as an activated thiolester from a Ub-activating enzyme (E1) to a Ub-conjugating enzyme (E2), and finally to the protein target with the help of a Ub protein ligase (E3) (1, 2).

The assembly of poly-Ub chains onto a targeted protein has proven to be a hallmark of a variety of processes, such as cell cycle control (3), DNA repair (4), ribosome biogenesis (5), the inflammatory response (6), endocytosis of cell surface proteins (7), and NF-kappa B-dependent signal transduction (8). These chains are synthesized in an E2-dependent reaction where each Ub within the chain is covalently bound to its neighbor by an isopeptide bond that links the C terminus to a surface lysine of its target-proximal Ub partner. Previous observations have demonstrated that these chains can exist in different configurations that are defined by the specific lysine residue that links each Ub molecule within the chain (9-14).

The most prevalent and best-documented examples of protein ubiquitination use the Lys-48-linked chain configuration to target proteins for degradation by the 26 S proteasome (2). Recently, however, a non-proteolytic ubiquitination pathway has come to light that results in the substrate-tethered assembly of multi-Ub chains, where Ub molecules are tandemly linked to one another through Lys-63 (12, 15-19). This pathway plays a key role in error-free DNA post-replicative repair (20-22), endocytosis (15), and polysome stability (17) and is an important component of NF-kappa B signal transduction (18, 19).

The error-free repair and NF-kappa B pathways both catalyze the assembly of Lys-63 chains using a conserved E2 heterodimer, composed of a catalytically active hUbc13 subunit and a catalytically inactive E2-like subunit termed ubiquitin-conjugating enzyme variant (UEV). UEV proteins share significant sequence similarity with other E2s but lack the characteristic active-site cysteine residue required for thiolester formation. In the error-free repair pathway of Saccharomyces cerevisiae, two chromatin-associated RING finger proteins, Rad5 and Rad18, recruit the Ubc13·Mms2 heterodimer and Ubc2 (Rad6) to DNA (23). In very recent work, Hoege et al. (24) have demonstrated that a target of this pathway is the yeast proliferating cell nuclear antigen, which is first mono-ubiquitinated through Rad6 and Rad18 and then poly-ubiquitinated by Ubc13·Mms2 in conjunction with Rad5. In NF-kappa B signal transduction, Traf6, a RING domain E3 protein, functions together with the hUbc13·UEV heterodimer (containing either hUEV1a or the functionally equivalent hMms2) in the formation of Lys-63-linked poly-Ub chains that are required for the activation of Ikappa B kinase, a key signal transducer in the NF-kappa B pathway (18, 19).

Insight into the mechanism of Lys-63 chain assembly and its relationship to structure recently became possible with the simultaneous determination of both S. cerevisiae (25) and human (26) Ubc13·Mms2 heterodimer structures by x-ray crystallography and an NMR-based approach for mapping the protein-protein interactions within the Ub-bound complex (27). Using the previously determined assignments for Ub in 1H-15N HSQC NMR experiments, we were able to footprint the surface of Ub that interacted with the human hUbc13·hMms2 heterodimer and each of its subunits in either the thiolester-linked or unlinked forms (27). The results of this study were consistent with a two-binding site model in which an "acceptor" molecule of Ub bound non-covalently to hMms2 was positioned in an orientation such that a second Ub molecule that was linked to hUbc13 as a thiolester could be transferred to Lys-63 of the accepting Ub molecule. The NMR assignments of both hMms2 and hUbc13 is an obvious prerequisite for footprinting the surfaces of the heterodimer that interact with both the covalently linked and unlinked forms of Ub. In the present work we have determined the footprint that both Ub molecules make on the surface of the hUbc13·hMms2 heterodimer. Taken together with our previous work, a compelling model is presented for the tetrameric structure that places Lys-63 of the accepting Ub molecule in catalytic proximity of the C terminus of the donor Ub molecule.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein Expression-- hUbc13 and hMms2 were expressed and purified as described previously (27) with the following exceptions. Proteins were expressed in the Escherichia coli strain BL21(DE3)-RP (Stratagene), and 2-liter cultures were grown at 25 °C to A590 = 0.3 in minimal media containing 15NH4Cl as the sole nitrogen source and induced with isopropyl-beta -D-thiogalactopyranoside (0.4 mM) for an additional 24 h at 25 °C. S. cerevisiae UbK48R, UbK63R, and Uba1 (E1) were expressed and purified as described previously (27).

NMR Spectroscopy-- All NMR spectra were obtained using a Varian Unity INOVA 600-MHz spectrometer at 30 °C. The two-dimensional 1H-15N-HSQC NMR spectra were acquired using the sensitivity-enhanced gradient pulse scheme developed by Kay and co-workers (28, 29). The 1H and 15N sweep widths were 8000 and 2200 Hz, respectively. A minimum of 64 transients was collected for each spectrum. All NMR samples were prepared to include HEPES (50 mM, pH 7.5), NaCl (75 mM), EDTA (1 mM), dithiothreitol (1 mM), and 2,2-dimethyl-2-silapentane-5-sulfonate (1 mM) in the presence of 9:1 H2O:D2O.

Spectral processing was accomplished with the NMRPipe program (30). The NMRview program (31) was employed in the assignment of all two-dimensional 1H-15N-HSQC NMR cross-peaks. To calculate the total average change in backbone amide 1HN and 15N chemical shifts for each resonance, the following equation was applied (32),
&Dgr;&dgr;<SUB><UP>total</UP></SUB>=<RAD><RCD>(&Dgr;&dgr;<SUP>15</SUP><UP>N</UP>)<SUP>2</SUP>+(&Dgr;&dgr;<SUP>1</SUP><UP>HN</UP>)<SUP>2</SUP></RCD></RAD> (Eq. 1)
where Delta delta 15N and Delta delta 1H are the chemical shift changes in hertz. The average change in total chemical shift was then calculated for each identified residue, with the exception of those whose resonances had broadened past detectability in the two-dimensional 1H-15N-HSQC NMR spectra. The standard deviation associated with each dataset was also calculated.

15N-hMms2 Chemical Shift Perturbation Experiments-- An initial two-dimensional 1H-15N-HSQC spectrum was acquired for 15N-hMms2 (250 µM) as a point of reference for subsequent chemical shift perturbation experiments. The spectrum also served to confirm the proper folding and lack of aggregation of 15N-hMms2.

The interactions between 15N-hMms2 and hUbc13 were examined by inclusion of a slight excess of unlabeled hUbc13 (300 µM) to the sample described above for 15N-hMms2 alone. The NMR tube was allowed to equilibrate for 1 h at 30 °C to ensure heterodimerization would proceed to completion. A two-dimensional 1H-15N-HSQC spectrum was then acquired for the sample.

Non-covalent interactions between 15N-hMms2 (250 µM) and Ub were examined by including unlabeled UbK48R (600 µM) into NMR samples in the presence or absence of unlabeled hUbc13 (300 µM). A two-dimensional 1H-15N-HSQC spectrum was then acquired for each sample. Chemical shift assignments in the two-dimensional 1H-15N-HSQC spectra were again completed assuming that the closest cross-peak represented the correct change in chemical shift. The two-dimensional 1H-15N-HSQC NMR reference spectrum used when calculating changes caused by Ub were either (i) 15N-hMms2 alone to examine the changes cause in hMms2 by itself or (ii) 15N-hMms2·hUbc13 to probe the changes in chemical shift in hMms2 in the context of the heterodimer.

15N-hUbc13 Chemical Shift Perturbation Experiments-- As in the case of hMms2, an initial two-dimensional 1H-15N-HSQC NMR spectrum was acquired as a point of reference and confirmed the proper folding and lack of aggregation of 15N-hUbc13 (305 µM).

The interactions between 15N-hUbc13 and hMms2 were examined by inclusion of a slight excess of unlabeled hMms2 (330 µM) to the sample described above for 15N-hUbc13 alone. Sample equilibration and acquisition were performed as described for the 15N-hMms2 samples.

Thiolester-linked interactions between 15N-hUbc13 (305 µM) and Ub (330 µM) were examined in situ by inclusion of S. cerevisiae E1 (0.3 µM), ATP (5 mM), and MgCl2 (5 mM) as described previously (27). Addition of hMms2 (330 µM) to this sample allowed for the examination of the hMms2·15N-hUbc13-Ub species. Studies described elsewhere (27) have shown that thiolester formation is rapid (minutes) whereas the formation of Ub conjugate on hUbc13 is slow (hours). Furthermore, the onset of conjugate formation can be clearly identified based on the accumulation of new peaks emanating from the mixed population of Ub species. The two-dimensional 1H-15N-HSQC NMR experiments were therefore performed between 10 and 120 min after the addition of E1 to minimize the impact of possible side-reactions. UbK63R was employed as the Ub species to eliminate the possibility of chain formation by the hUbc13·hMms2 heterodimer and, hence, to eliminate further complication of the spectra (27). The two-dimensional 1H-15N-HSQC NMR reference spectra used when calculating changes caused by Ub in thiolester complexes were either (i) 15N-hUbc13 alone to examine the changes caused in hUbc13 by itself or (ii) 15N-hUbc13·hMms2 to probe the changes in chemical shift in hUbc13 in the context of the heterodimer.

Non-covalent interactions between 15N-hUbc13 and Ub were detected by including unlabeled UbK63R into NMR samples in the presence or absence of unlabeled hUbc13 under conditions identical to thiolester formation with the exception that E1, ATP, and MgCl2 were omitted. A two-dimensional 1H-15N-HSQC NMR spectrum was then acquired for each sample. However, no changes in 15N-hUbc13 cross-peaks were observed in either case, and therefore no further analysis was performed.

Molecular Modeling-- Molecular modeling of the surfaces of interaction was accomplished using the BiGGER soft-docking algorithm (33, 34) using the unbound structures of Ub (target) (35) and the hUbc13·hMms2 heterodimer (probe) (26). The BiGGER algorithm systematically searches the complete six-dimensional binding spaces of both target and probe and then evaluates these solutions in terms of a global scoring function consisting of geometric complementarity, electrostatic interactions, desolvation energy, and the pairwise propensities of amino acid side chains to interact across molecular interfaces. Docking parameters in this initial search included a 15° angular step, 5000 maximum solutions, and 300 minimum atomic contacts. The top 5000 solutions based on global score were then filtered using the NMR chemical shift perturbation data in the following manner. First, surface-exposed residues on hMms2 and Ub, respectively, which produced significant Delta delta total values upon non-covalent interaction were determined, and the number of atomic contacts between these two groups within a 5-Å distance cutoff in each of the top 5000 solutions as determined by global score was evaluated. The top solution based on these criteria was then accepted as the "correct" orientation and subsequently underwent minimization using the INSIGHTII suite of programs. The thiolester-bound Ub placement upon the heterodimer was then determined in an identical manner using Delta delta total values.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

When engaged in catalysis, the hUbc13·hMms2 heterodimer necessarily exists as part of a tetramer that is composed of the heterodimer in association with two Ub molecules. One Ub molecule is linked as a thiolester to the active site of hUbc13 (the donor) while the other Ub molecule interacts non-covalently with hMms2 (the acceptor). Although a high resolution crystallographic structure for the heterodimer has been determined (26), a crystallographic structure for the Ub-bound tetramer is unlikely. This conclusion is based both on the instability of the hUbc13-Ub thiolester bond (36) and the relatively weak interaction that exists between the acceptor Ub and hMms2 (Kd ~ 100 µM).2 Based on these considerations, we have pursued an alternative NMR-based approach to determine the structure of the hUbc13·hMms2-Ub2 tetramer. The tetramer has three major protein-protein interfaces: 1) the hMms2-hUbc13 interface, 2) the hMms2-Ub (acceptor) interface and, 3) the hUbc13-Ub (donor) interface. In this and previous studies (27), we have used 1H-15N HSQC NMR spectroscopy to observe the chemical shift perturbations that are induced upon interaction to define the footprint that each protein makes with its partner.

The method that we have chosen here relies upon the comparison of 1H-15N HSQC NMR spectra for each protein component in an unbound form and bound to its partner. To simplify the analysis, only one component of the complex is 15N-labeled in any given experiment. Backbone amide 1HN and 15N chemical shifts are sensitive to a variety of factors, including hydrogen bonding, electrostatic interactions, and aromatic ring current effects, to name a few. Therefore, changes to chemical shifts that can result from differences in chemical environment upon complex formation can be used to identify residues that are either directly involved at the binding interface or correspond to long range structural changes.

A necessary precursor to chemical shift mapping is the complete assignment of backbone amide 1HN-15N cross-peaks in the 1H-15N HSQC NMR spectra for a given component of the complex. Recently, we have completed the full backbone chemical shift assignments for both hUbc13 and hMms2 (available upon request). Each protein exhibits well dispersed and resolved 1H-15N HSQC NMR spectra at 600 MHz, as is shown for hMms2 (Fig. 1). Furthermore, the spectra retain these qualities fairly well upon formation of higher order complexes of up to 42.5 kDa, although the signal-to-noise ratio is reduced as expected, due to increased linewidths. Chemical shift assignments in the two-dimensional 1H-15N-HSQC NMR spectra were made relative to the appropriate reference spectrum, assuming that the closest shifted cross-peak represented the correct one. This approach was required due primarily to the lability of complexes containing thiolester linkages.


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Fig. 1.   Superposition of 1H-15N HSQC NMR spectra of 15N-labeled hMms2, free and in complex with Ub. 1H-15N HSQC NMR spectra resulting from either 15N-hMms2 (black) or 15N-hMms2 and Ub (red) are overlaid, and a number of representative backbone cross-peaks, which were affected by complex formation, are labeled.

Mapping the Heterodimer Interface-- To map the interface between hUbc13 and hMms2, two heterodimer complexes were prepared in situ: one containing 15N-hUbc13 with unlabeled hMms2, and the other containing 15N-hMms2 with unlabeled hUbc13. The hUbc13·hMms2 heterodimerization (34 kDa) proceeds efficiently upon equimolar addition of each protein and results in the formation of a stable complex that remains associated during high resolution size-exclusion chromatography (27). Residues whose backbone amide 1H and 15N chemical shifts exhibited a perturbation upon complex formation were identified and quantified in terms of the total change in chemical shift, Delta delta total. The major Delta delta total values upon heterodimerization for either 15N-hMms2 or 15N-hUbc13 are associated with residues found at the heterodimer interface (Figs. 2B and 3B), indicating the similarity of this interface in both the crystalline and solution phases.


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Fig. 2.   Binding-induced NMR chemical shift perturbation analysis of hMms2 with Ub. Comparison of backbone amide 1H and 15N chemical shift of hMms2 in the absence or presence of Ub (A) and hUbc13 (B) or the comparison between 15N-hMms2·hUbc13 heterodimer and this heterodimer in the presence of Ub (C). The total change in chemical shift, Delta delta total, was calculated for hMms2 interacting with various binding partners and plotted as a function of primary amino acid sequence. The dashed lines represent the average change in Delta delta total and one standard deviation unit above this average. Residues whose change in chemical shift could not be identified are indicated with an asterisk.


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Fig. 3.   Binding-induced NMR chemical shift perturbation analysis of hUbc13 with Ub. Comparison of backbone amide 1H and 15N chemical shift of hUbc13 in the absence or presence of thiolester-linked Ub (A) and hMms2 (B) or the comparison between hMms2·15N-hUbc13 heterodimer and this heterodimer in the presence of thiolester-linked Ub (C). The total change in chemical shift, Delta delta total, was calculated for hUbc13 under each of the conditions and plotted as a function of primary amino acid sequence. Dashed lines represent the average change in Delta delta total as well as one standard deviation above this average. Residues whose change in chemical shift could not be identified are indicated with an asterisk.

Residues resulting in the greatest effect on Delta delta total for interactions within the heterodimer or between the heterodimer and Ub (see below) have been summarized in Fig. 4 according to sequence and secondary structure. Also shown are the chemical shift indices for each residue contained in hMms2 and hUbc13, which provide a measure of the deviation between the observed chemical shifts and their random coil values, and are indicative of the type of secondary structure (37, 38). A comparison between secondary structural elements for hMms2 and hUbc13, determined by x-ray crystallography to those determined from the chemical shift indices, demonstrate a close correlation between types of secondary structure determined in the solution and crystalline states.


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Fig. 4.   Sequence alignments of the important interfacial residues in hUbc13 and hMms2 with S. cerevisiae Ubc1 as determined by 1H-15N HSQC NMR chemical shift perturbation. Residues experiencing the greatest Delta delta total upon formation of hMms2·hUbc13 are colored in yellow and blue, respectively, and are compared with interfacial residues in the crystal structure (boxed) (26). hMms2 residues experiencing the most significant Delta delta total upon formation of non-covalent interaction with Ub are labeled in red, as are residues in hUbc13 upon formation of the thiolester adduct with Ub. For comparison, residues deemed responsible for the interaction between Ubc1 and Ub in the thiolester complex are also colored red (40). Secondary structural elements are shown above (for E2s) and below (for hMms2) the sequence alignments, as are the average chemical shift index values as determined from Ca, Co, and Ha chemical shifts (up arrow = +1, down arrow = -1, no arrow = 0) as obtained from the program NMRview program using the Wishart peptide data base (38), pH 7.5, and 303 K.

The Non-covalent Interaction between hMms2 and Ub-- Both hMms2 and hUbc13 have each been observed to exist in a monomeric state and as the heterodimer (23, 27), whereas homodimerization has not been observed (see "Experimental Procedures"), and therefore an examination of the interaction between Ub and the hMms2 subunit is of interest. The chemical shift perturbations that result from the interaction of 15N-hMms2 subunit with unlabeled acceptor Ub are shown in Fig. 2A. The greatest effects on Delta delta total upon interaction with Ub are observed at the N-terminal portion of hMms2. Specifically, the affected residues are located in helix alpha 1 (Glu-20, Gly-22, Lys-24), sections of strand beta 1 (Val-31, Ser-32, Leu-35), strand beta 2 (Thr-47, Gly-48, Met-49), strand beta 3 (Tyr-63, Leu-65), helix alpha 2 (Leu-119) as well as the loop joining helix alpha 1 to strand beta 1 (Val-26, Thr-30). Intermediate effects on Delta delta total are found close in sequence to the greatest changes and include the C-terminal portion of alpha 1 (Gln-23), sections of beta 1 (Trp-33), beta 2 (Trp-46), L2 prior to beta 3 (Asn-60, Arg-61), beta 3 (Val-67, Gly-70), and the loop joining alpha 1 to beta 1 (Gly-25, Gly-27, Gly-29). Intermediate changes are also found in alpha 2 (Gln-120, Leu-125, Glu-130) and the C terminus (Gly-140, Gln-141).

As expected, many of the residues in hMms2 that exhibit the greatest backbone amide chemical shift perturbations are located on the surface of the protein, and contain surface exposed side chains that may be involved in non-covalent interactions with Ub (Fig. 5A). These residues cluster onto one face of hMms2, forming three distinct patches. Interestingly, no significant changes in chemical shift were observed for residues on the opposite surface of hMms2. The first patch is perpendicular to the hUbc13·hMms2 interface, and is composed of residues at the C-terminal end of alpha 1 and the loop that joins alpha 1 to beta 1 (Glu-20, Glu-21, Gly-22, Gln-23, Lys-24, Gly-25, Val-26, Gly-27, Gly-29, and Val-31), portions of beta 1 (Ser-32, Trp-33, and Leu-35), beta 2 (Thr-47, Gly-48, and Met-49), and beta 3 (Arg-61, Tyr-63, and Leu-65). The second patch is found at the C-terminal portion of hMms2. Notably, the total surface area of both these hMms2 patches corresponds well with the complementary patch on Ub that has previously been demonstrated to interact with hMms2 (27). Additionally, the combined electrostatic surface potential of the hMms2 patches is complementary to that found on Ub (Fig. 5C). Interestingly, the third patch involves hMms2 residues that would normally interact with hUbc13 in the heterodimer, and include Val-7, Lys-8 (greatest Delta delta total), and other N-terminal amino acids of hMms2 (intermediate Delta delta total).


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Fig. 5.   Connolly surfaces of the binding interfaces on hMms2 or hUbc13 upon interaction with Ub. The surface of hMms2 is presented either alone (A) or in the context of hMms2· hUbc13 heterodimer (hUbc13, yellow) (B). The surface of hUbc13 is presented either alone (D) or in the context of hMms2· hUbc13 heterodimer (hMms2, blue) (E). Residues affected by non-covalent interaction with Ub are colored with a linear gradient from white (Delta delta total = 0) to dark red (Delta delta total >=  Delta delta total(av)+1s) as determined by 1H-15N HSQC NMR chemical shift perturbation analysis (Figs. 2 and 3). Residues, whose Delta delta total could not be determined unambiguously due to broadening or extreme changes in chemical shift are colored orange. The active-site cysteine (Cys-87) of hUbc13 is colored green as a point of reference. Electrostatic surface potential of the hMms2·hUbc13 heterodimer (C and F) is shown in the same orientation as B and E, respectively. The relative electrostatic potentials are displayed as a linear gradient, from acidic (-10, red), to neutral (0, white), to basic (+10, blue) as determined by the program GRASP (49).

Our previous findings indicated that the Ub contact surface with hMms2 remained largely the same when alone or in complex with hUbc13 (27). When we next examined the 15N-hMms2-Ub interaction as a heterodimer with hUbc13 we similarly found that the hMms2 residues that undergo change on Ub binding closely parallel those of the individual subunit with some notable exceptions (Fig. 2C). As with hMms2 alone, many of the major Delta delta total are found near the C terminus of alpha 1 (Glu-20, Gln-23), the loop that joins it to beta 1 (Val-26, Gly-29, Thr-30), beta 1 (Val-31), beta 2 (Gly-48, Met-49), and beta 3 (Arg-61, Tyr-63, Leu-65). Residues with intermediate values of Delta delta total are also similar, including alpha 1 (Leu-19, Glu-21), the loop joining alpha 1 to beta 1 (Gly-25), beta 1 (Trp-33, Gly-34), beta 2 (Thr-47, Gly-52), beta 3 (Asn-60, Ile-62, Val-67), alpha 2 (Ser-114, Ile-115, Val-117, Gln-120, Leu-125, Glu-130), and the C terminus (Gln-141). The backbone amide 1HN-15N HSQC NMR cross-peaks for three residues (L1 (Asp-37) and beta 2 (Arg-45, Ile-50)) either experienced large changes in chemical shift, rendering identification difficult, or their intensities were severely diminished due to line-broadening as a result of complex formation.

In contrast to the hMms2 subunit alone, none of the N-terminal residues situated at the heterodimer interface undergo significant change upon Ub binding, whereas significant change is detected within L1 (Asp-38, Asp-40, Met-41, and Arg-45). Notably, the region surrounding the vestigial active site of hMms2 does not appear to play a role in Ub binding. This result clearly distinguishes the hMms2-Ub interaction from other previously reported E2-Ub interactions. The changes in the surface characteristics of the hMms2 component of the heterodimer upon Ub binding are shown in Fig. 5B.

The Interaction between hUbc13 and Thiolester-linked Ub-- The major changes to the 15N-hUbc13 subunit that result from thiolester formation with Ub are found in and around the active-site (Cys-87) (Fig. 3A). These include: the active-site cysteine itself, L4 (Asn-79, Leu-83, Arg-85) to the N-terminal side of Cys-87, the 3-10 helix C-terminal to Cys-87 (Asp-89, Ile-90), the loop preceding helix alpha 3 (Leu-111, Asn-116, Asp-118, Asp-119), and helix alpha 3 (Asp-124, Val-125, Glu-127, Lys-130). Intermediate perturbations of Delta delta total are found around and inter-digitated with the major changes described above. These include: L4 (Met-72, Ile-75, Tyr-76, His-77), near the active site (Leu-88), the 3-10 helix (Lys-92, Trp-95, Ser-96, Ala-98), the loop preceding alpha 3 (Ser-113, Ala-114), and alpha 3 (Ala-126, Thr-131).

Heterodimerization of 15N-hUbc13 with hMms2 results in somewhat fewer Delta delta total upon thiolester formation when compared with the thiolester formed with 15N-hUbc13 alone (Fig. 3, when comparing C with A). It is noted, however, that a number of cross-peaks in the 1H-15N HSQC NMR spectra of the heterodimer thiolester remain unassigned due to line broadening or large changes in chemical shift upon complex formation. The major and intermediate changes to Delta delta total occur within secondary structural regions, including L4 (Lys-74, Ile-75, Tyr-76, Asn-79, Leu-83, Gly-84, and Arg-85), the active-site (Cys-87), the 3-10 helix (Leu-88, Asp-89, Ile-90, Leu-91, and Asp-93), the loop preceding alpha 3 helix (Asn-116, Asp-118, Leu-121, Ala-122, and Asp-124), and the alpha 3 helix (Val-125, Ala-126, Trp-129, K130, and Thr-131).

Surfaces involved in the interaction between hUbc13 and its thiolester-linked Ub were determined by mapping the major Delta delta total for the 15N-hUbc13 subunit alone or in complex with hMm2s onto a surface projection of the hUbc13 crystal structure (Fig. 5, D and E). In the absence of hMms2 (Fig. 5D), the greatest effect is found around the active site (Cys-87) where the majority of affected residues have solvent-exposed side chains (L4: Arg-70, Leu-83, Arg-85, Ile-86, Cys-87, and Asp-89; alpha 2: Leu-106, Gln-109, Ala-110, and Leu-111; alpha 3 and preceding loop: Asn-116, Asp-118, Asp-119, Asp-124, Ala-126, Glu-127, and Lys-130).

From Fig. 5E, it is apparent that hUbc13 exhibits a similar Ub-dependent pattern of backbone amide chemical shift changes when present with hMms2. Significantly, all of the solvent-exposed residues important in thiolester formation present themselves on only one face of the hUbc13 molecule regardless of dimerization state. We conclude from these results that the hUbc13-Ub thiolester interaction is largely unaffected by the presence or absence of hMms2. These results are consistent with our previous NMR experiments demonstrating that both the C-terminal tail and a slightly basic surface on Ub form contacts with hUbc13 within the hUbc13-Ub thiolester regardless of the presence of hMms2 (27).

Modeling the Tetramer-- The soft-docking algorithm BiGGER (33, 34) was employed to generate models for the Ub2-hUbc13-hMms2 tetramer based on geometric complementarity, electrostatic interactions, desolvation energy, and the pairwise propensities of amino acid side chains to interact across interfaces. Surface residues from the heterodimer (results presented herein) and Ub (27), which exhibited the greatest change toDelta delta total upon complex formation, were incorporated as constraints into the BiGGER docking program (see "Experimental Procedures"). The C terminus of the donor Ub was not covalently linked to the active site of hUbc13. The top ten structures based on these criteria were subsequently averaged, and the resulting structure was subjected to energy minimization using the INSIGHTII suite of programs. The final structure of the model is shown in Fig. 6.


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Fig. 6.   NMR-derived model of the tetrameric Ub-conjugating enzyme complex. A, the surfaces of interaction between either acceptor (top) or donor (bottom) Ub molecules (red, ribbon) and the hUbc13 (yellow)/hMms2 (blue) heterodimer are presented. Of specific interest is the active-site Cys-87 of hUbc13 (green), Lys-63 of the acceptor Ub (purple), and Gly-76 of the donor Ub (purple). Residues hypothesized to represent the RING binding domain are white. The NMR-derived model of the tetrameric complex was determined using the BiGGER docking algorithm (33, 34) and the INSIGHTII suite of programs as described under "Experimental Procedures." B, close-up of the model of the region surrounding Cys-87 of hUbc13.

The non-covalent interaction between acceptor Ub and the heterodimer involves hydrophobic contacts between Ub and hMms2. The surface-exposed residues of the beta -sheet of the acceptor Ub, and the loops connecting strands within the sheet, constitute the contact interface with hMms2, whereas hMms2 residues that contact the acceptor Ub are found in alpha 1, beta 1, and beta 2 and the loops connecting these secondary structural elements. The hMms2 surface involved in the interaction is located opposite to the surface containing the vestigial active site. The donor Ub makes contacts with hUbc13 through C-terminal residues 70-76, as well as some residues in beta 1 and beta 3. The hUbc13 residues that form contacts with the C terminus of donor Ub are found within the active site, the loops preceding it, and residues in alpha 2.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Together, the NMR chemical shift perturbation results have been interpreted to produce a model of the tetramer using a molecular docking strategy that is tailored to this NMR-based approach. The accepting Ub molecule sits on a concave face of hMms2, a distinctive feature of both E2s and UEVs, with its C-terminal tail far removed from the vestigial active site of hMms2. In combination with hUbc13, the concave face of hMms2 narrows to form a channel or funnel as it approaches the active site of hUbc13. The side-chain Lys-63 for the acceptor Ub lies within this channel, placing the epsilon -nitrogen within 3 Å of the sulfur atom contained within the active-site cysteine of hUbc13. The interaction between the accepting Ub and the heterodimer buries 2792 Å2 of surface area, a rather large value in light of our observation that the interaction between the two is weak (Kd ~ 100 µM).2 The model likely overestimates the buried surface area of the acceptor Ub, because the imposed chemical shift restraints force the contact regions to be maximized and may include residues that are affected indirectly through induced structural changes in the proteins.

There are two features of the accepting Ub-heterodimer interface that bear directly on its biochemical function. First, the C-terminal tail of the acceptor is neither constrained nor sterically hindered, raising the likelihood that it can serve as the poly-Ub chain anchor in either the free form or when attached to an appropriate protein target. Second, Lys-48 of the acceptor is buried within the protein-protein interface, thereby excluding this residue as a potential site for chain assembly of the canonical type.

The donor Ub interacts exclusively with a hydrophobic concave surface that narrows to an acidic cleft on hUbc13 and culminating with the active site cysteine (Fig. 5F). The tail of the donor Ub lies within the active site cleft of the E2 placing the C-terminal carboxyl carbon of Gly-76, the active site sulfur and the epsilon -nitrogen of Lys-63 for the acceptor Ub molecule within 3.5 Å of each other.

In terms of the position and orientation of the components, the model presented here agrees moderately well with that proposed by VanDemark et al. (25) for the S. cerevisiae complex. It differs significantly, however, from the model proposed by Pornillos et al. (39) who examined the non-covalent interaction between the human Tsg101 UEV domain and Ub by a similar approach to the one used here. The structural differences between the Ub-hMms2 interaction and Ub-Tsg101 interaction results from the presence of an extended beta -hairpin that links strands 1 and 2 in Tsg101 that sequester Ub. The fact that this motif is absent in hMms2 illustrates that UEVs have evolved different strategies for Ub binding.

Our high confidence in this model stems from the NMR-constrained docking approach used here. The docking algorithm BiGGER is particularly well suited for these analyses because of its ability to use NMR chemical shift perturbation results as information to filter suitable models (33, 34). The BiGGER docking algorithm requires no information that constrains the orientation of the docking partners and, therefore, represents a fairly unbiased approach for using NMR data to model the tetramer interactions. The validation of this approach lies in the predicted positions of the three atoms involved in linking the C terminus of the donor Ub molecule to Lys-63 of the accepting Ub molecule: 1) the cysteine sulfur atom of the hUbc13 active site, 2) the Gly-76 carboxyl group of the Ub donor molecule, and 3) the Lys-63 epsilon -nitrogen of the accepting Ub molecule. Each of these atoms is positioned within 3.5 Å of each other (Fig. 6).

The model presented here also agrees well with the findings of a previous mutagenesis study that used the S. cerevisiae Ubc13·Mms2 heterodimer (25). A Ubc13 substitution (A110R) located on the surface of alpha 3, near the center of the predicted interaction between Ubc13 and the donor Ub, resulted in a 4-fold reduction in the rate of isopeptide bond formation. A Ubc13 substitution (D81A) situated nearby the predicted position of Lys-63 of the accepting Ub resulted in a diminished affinity of the acceptor Ub for the heterodimer in vitro. A Ub substitution (I44A) located in the NMR-derived surface for the acceptor but not donor, results in reduced binding of Ub to the acceptor site on Mms2, whereas the interaction with Ubc13 remains unaffected. Conversely, an Mms2 substitution (E12R) situated near the heterodimer interface but not predicted by the model to play a role in acceptor Ub binding does not weaken the interaction of the acceptor Ub with the heterodimer in vitro (25).

The structure of the hUbc13-Ub thiolester presented here holds features in common with the models for the Ubc1-Ub thiolester from S. cerevisiae (40) and the human Ubc2b-Ub serine ester (36), each derived by similar NMR-based approaches. All three E2s employ a common thiolester-binding motif (L4 around the active site, regions of alpha 2, and the loop that joins alpha 2 to alpha 3) that constrains the C-terminal tail similarly among models. In contrast, the folded domain of Ub is positioned slightly differently on the each of the three E2s (Fig. 4). These differences are likely explained by properties associated with catalysis. The tail of the Ub donor must be bound to the E2 strongly enough to secure its alignment during isopeptide bond formation with the target, yet weakly enough to assure efficient transfer and subsequent turnover of the E2. E2 interactions with the rest of the Ub globular domain are therefore likely to be even weaker and can be imagined to vary significantly by differences of a few key surface residues from one E2 to the next.

An examination of high resolution E2 structures has revealed that the active site is part of an unstructured loop (41-47). Our previous and present findings suggest that the interaction of hMms2 with hUbc13 alters the activity of hUbc13 by altering the conformation of the hUbc13 active site. We have previously shown that when hMms2 binds to hUbc13, both the rate of Ub thiolester formation with hUbc13 (reduced 2-fold in the presence of hMms2) and the stability of the resulting thiolester are measurably affected in vitro (27). This observation raises the intriguing possibility that the interaction of an E2 with other proteins could order the loop in a particular conformation, thereby modulating its catalytic activity.

An examination of the chemical shift perturbation data reveals that there is communication between the acceptor and donor Ub binding sites. This is reflected by a change in chemical environment at residues that are known to play a key role in the active-site loop. For instance, residues in the active-site cleft of hUbc13 (Leu-83, Gly-84, Arg-85, Leu-88, and Ile-90) show significant values of Delta delta total upon dimerization with hMms2. Three of these residues (Leu-83, Gly-84, and Arg-85) are directly involved in the heterodimer interface, whereas two of these residues (Leu-88 and Ile-90) are remote from the interface. In addition, Ub thiolester formation within the heterodimer results in a significant shift of Delta delta total for the interfacial residues Leu-83 and Arg-85. This observation suggests that the communication between the heterodimer interface and the active site is in fact occurring, that is, altering the interface alters the active site and vice versa. These results appear to be in contrast with those previously reported for S. cerevisiae Ubc13·Mms2, for which there appears to be little communication between the dimer interface and the active site. An r.m.s.d. of 0.8 Å for superimposition of all backbone Calpha atoms between free and Mms2-bound Ubc13 was reported, with the active site cleft little changed (25). However, as chemical shift changes cannot be directly converted into three-dimensional structural changes, further analyses will be required to establish the extent of similarities and differences between the human and S. cerevisiae protein complexes.

The arrangement of the four molecules within the tetramer poses no obvious steric problem for the interaction of hUbc13 with its functionally specific E3, Traf6. The interface between hUbc13 and Traf6 can be predicted on the basis of the x-ray crystallographic structure for the E2·E3 complex UbcH7·c-Cbl (48). Both c-Cbl and Traf6 contain E2-binding RING finger domains that share significant sequence identity. Traf6 likely sits on an 11-residue patch of hUbc13, with six residues identical to those employed by UbcH7 in its interaction with c-Cbl (Fig. 6). Notably, none of these residues are involved in forming contacts between Ub and hUbc13.

Despite its small size and highly conserved fold, the E2 core domain family is apparently the centerpiece for several distinct biochemical functions that hinge on isopeptide bond formation. These functions include both target ubiquitination and the synthesis of multi-Ub chains that differ from one another in configuration. As a consequence of unknown evolutionary pressure, these proteins have apparently modeled and remodeled their surfaces with great economy and creativity. The functional repertoire of protein ubiquitination has been expanded by the ability of these proteins to interact with common or related partners in fundamentally different ways. This point is underscored in part by the present work. The E2 core fold has evolved at least three relevant and fundamentally different modes of Ub binding. Furthermore, the juxtaposition of two of these modes, through the interaction of a catalytically active fold with an inactive fold, provides the structural basis for Lys-63 multi-Ub chain synthesis.

    ACKNOWLEDGEMENTS

We thank Susan Smith for secretarial assistance, Linda Saltibus for technical assistance and all of the members of the Ellison and Spyracopoulos laboratories as well as Pascal Mercier and Prof. Brian Sykes for valuable input and assistance. We also thank Prof. Lewis E. Kay for pulse sequences, Deryck Webb for spectrometer maintenance, and Yanni Batsiolas and Robert Boyko for computer expertise.

    FOOTNOTES

* This work was supported in part by research grants from the National Cancer Institute of Canada (to M. J. E.) and the Alberta Heritage Foundation for Medical Research (AHFMR) (to L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The online version of this article (available at http://www.jbc.org) contains Supplemental Figs. 1 and 2.

§ A Natural Sciences and Engineering Research Council of Canada scholar.

A Canadian Institutes of Health Research (CIHR) scholar.

** Funded by CIHR (Grant MOP-53240).

§§ An AHFMR medical scholar. To whom correspondence may be addressed: Dept. of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-2417; Fax: 780-492-0886; E-mail: leo.spyracopoulos@ualberta.ca.

¶¶ To whom correspondence may be addressed: Dept. of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-6352; Fax: 780-492-0886; E-mail: mike.ellison@ualberta.ca.

Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M212353200

2 S. McKenna, J. Hu, T. Moraes, W. Xiao, L. Spyracopoulos, and M. J. Ellison, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: Ub, ubiquitin; UEV, ubiquitin-conjugating enzyme variant; HSQC, heteronuclear single quantum coherence; E1, Ub-activating enzyme; E2, Ub-conjugating enzyme; E3, Ub protein ligase.

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RESULTS
DISCUSSION
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