Volume 21, Issue 6, Pages 873-880 (March 2006) A UbcH5/Ubiquitin Noncovalent Complex Is Required for Processive BRCA1-Directed Ubiquitination  Peter S. Brzovic, Alexei Lissounov, Devin E. Christensen, David W. Hoyt, Rachel E. Klevit  Molecular Cell  Volume 21, Issue 6, Pages 873-880 (March 2006) DOI: 10.1016/j.molcel.2006.02.008 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Structure of the UbcH5c/Ub Noncovalent Complex (A) Overlay of an expanded region of two-dimensional (2D) 1H-15N HSQC spectra collected at 600 MHz of 0.4 mM 15N labeled UbcH5c with 0 (black spectrum) and 2.0 equivalents (red spectrum) unlabeled Ub. (B) Ribbon representation of the UbcH5c (gray)/Ub (red) complex structure. Cys 85, in the E2 active site, is shown in yellow. (C) Side chain interactions at the binding interface. The structure of UbcH5c is rotated by 70° on the long axis relative to the view shown in (B). Ub side chains at the binding interface are shown in red; UbcH5c residues are shown in green. (D) UbcH5c (black) and Ub (red) residues whose side chains give reciprocal intermolecular NOEs in 13C F1-filtered, F3-edited NOESY spectra collected at 800 MHz. Molecular Cell 2006 21, 873-880DOI: (10.1016/j.molcel.2006.02.008) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Western Analysis of BRCA1-Directed Ubiquitination Reactions Reactions were performed with FLAG-tagged BRCA1 and T7-tagged Ub and time points analyzed by SDS-PAGE. The same gel was probed simultaneously with (A) anti-FLAG to visualize FLAG-BRCA1 and FLAG-BRCA1-ubiquitinated products and (B) anti-T7 to visualize all ubiquitinated products. Lanes 1–5, reaction using wt-UbcH5c at 0, 5, 10, 20, and 60 min after addition of ATP; lane 6, molecular weight standards (BioRAD Precision Plus); and lanes 7–11, reaction with S22R-UbcH5c at 0, 5, 10, 20, and 60 min after addition of ATP. Molecular Cell 2006 21, 873-880DOI: (10.1016/j.molcel.2006.02.008) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Spectra of Thiolester UbcH5c∼Ub Complexes (A) 1H-15N HSQC spectral overlay for a conjugation reaction coupling unlabeled Ub to 15N-S22R-UbcH5c before (black) and ∼2 hr after (red) addition of ATP. A subset of resonances that shift upon thiolester formation are labeled. (B) Same reaction as in (A) except with 15N-Ub and unlabeled S22R-UbcH5c. Overlay of 1H-15N HSQC spectra before (black) and ∼2 hr after (red) addition of ATP. Ub residues 73–76, the only resonances affected, are labeled. (C) UbcH5c and Ub residues whose amide resonances are perturbed upon thiolester formation (colored red) mapped onto ribbon representations of their respective structures. (D) Same reaction as shown in (A) except with wt-15N-UbcH5c. The 1H-15N HSQC spectrum shown was collected ∼2 hr after addition of ATP. Almost all resonances are severely reduced in intensity. The peaks located from 6.5 to 7.6 ppm (HN) and 110 to 113 ppm (N) represent a subset of Asn and Gln side chain amide resonances exposed on the surface of UbcH5c. The 1H-15N HSQC spectrum of the reaction mixture prior to the addition of ATP (data not shown) is identical to the spectrum of the UbcH5c/Ub noncovalent complex shown in Figure 1A. Molecular Cell 2006 21, 873-880DOI: (10.1016/j.molcel.2006.02.008) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Self-Assembly of UbcH5c∼Ub (A) Gel-filtration profiles of conjugated UbcH5c∼Ub species. Two-hundred micromolar samples of wt-UbcH5c∼Ub or S22R-UbcH5c∼Ub were chromatographed on an analytical Superdex 75 10/30 column. Wt-UbcH5c∼Ub (black trace) elutes earlier than S22R-UbcH5c∼Ub (gray trace), indicating wt-UbcH5c∼Ub forms higher molecular weight complexes. The elution profile of wt-UbcH5c∼Ub, unlike S22R-UbcH5c∼Ub, is dependent on sample concentration (see text). (B) Cartoon depicting self-assembly of activated wt-UbcH5c∼Ub. The I44 surface of conjugated Ub is exposed and available to bind noncovalently to another wt-UbcH5c∼Ub molecule. Self-assembly can continue in either direction. Although intermolecular interactions are not observed in solution for unconjugated UbcH5c, self-assembly of activated UbcH5∼Ub may promote such contacts. As shown in Figure 1, noncovalent binding of Ub to UbcH5c positions the C termini of both proteins adjacent to one another. In a self-assembling complex, binding of a conjugated Ub to a noncovalent site on another UbcH5c would bring two molecules of Ub-conjugated UbcH5c into close proximity, thereby increasing the potential for intermolecular UbcH5c-UbcH5c interactions. Minor structural changes that may occur in UbcH5 as a consequence of conjugation with Ub may promote such contacts. Because the noncovalent Ub binding site does not overlap with the E3 binding surface (Figure S2), UbcH5c can simultaneously interact with both Ub and an E3 Ub ligase. Molecular Cell 2006 21, 873-880DOI: (10.1016/j.molcel.2006.02.008) Copyright © 2006 Elsevier Inc. Terms and Conditions