The conjugation of ubiquitin to proteins involves a cascade of activating (E1), conjugating (E2), and ubiquitin-ligating (E3) type enzymes that commonly signal protein destruction. In TGFbeta signaling the inhibitory protein Smad7 recruits Smurf2, an E3 of the C2-WW-HECT domain class, to the TGFbeta receptor complex to facilitate receptor degradation. Here, we demonstrate that the amino-terminal domain (NTD) of Smad7 stimulates Smurf activity by recruiting the E2, UbcH7, to the HECT domain. A 2.1 A resolution X-ray crystal structure of the Smurf2 HECT domain reveals that it has a suboptimal E2 binding pocket that could be optimized by mutagenesis to generate a HECT domain that functions independently of Smad7 and potently inhibits TGFbeta signaling. Thus, E2 enzyme recognition by an E3 HECT enzyme is not constitutively competent and provides a point of control for regulating the ubiquitin ligase activity through the action of auxiliary proteins.

The RNA degradosome is a multiprotein complex required for the degradation of highly structured RNAs. We have developed a method for reconstituting a minimal degradosome from purified proteins. Our results demonstrate that a degradosome-like complex containing RNase E, PNPase, and RhlB can form spontaneously in vitro in the absence of all other cellular components. Moreover, ATP-dependent degradation of the malEF REP RNA by the reconstituted, minimal degradosome is indistinguishable from that of degradosomes isolated from whole cells. The Rne protein serves as an essential scaffold in the reconstitution process; however, RNase E activity is not required. Rather, Rne coordinates the activation of RhlB dependent on a 3 single-stranded extension on RNA substrates. A model for degradosome-mediated degradation of structured RNA is presented with its implications for mRNA decay in Escherichia coli.

Mono-and polyubiquitylation of proteins are key steps in a wide range of biological processes. However, the molecular mechanisms that mediate these different events are poorly understood. Here, we employed NMR spectroscopy to map a non-covalent ubiquitin binding surface (UBS) on the Smurf ubiquitin ligase HECT domain. Analysis of mutants of the HECT UBS reveal that interfering with the UBS surface blocked Smurf-dependent degradation of its substrate RhoA in cells. In vitro analysis revealed that the UBS was not required for UbcH7-dependent charging of the HECT catalytic cysteine. Surprisingly, although the UBS was required for polyubiquitylation of both Smurf itself and the Smurf substrate RhoA, it was not required for monoubiquitylation. Furthermore, we show that mutating the UBS interfered with efficient binding of a monoubiquitylated form of RhoA to the Smurf HECT domain. Our findings suggest the UBS promotes polyubiquitylation by stabilizing ubiquitylated substrate binding to the HECT domain.Ubiquitylation of protein substrates via an E1-E2-E3 enzymatic cascade is important for many biological processes. There is a panoply of ubiquitin modifications that can regulate the outcome of ubiquitylation, in particular, chain length. For example, monoubiquitylation is critical for directing trafficking of proteins through the endosomal system, whereas polyubiquitylation plays a key role in directing substrates to the proteasome for degradation (1). One class of E3 ubiquitin ligases is HECT domain ubiquitin ligases that can both mono-and polyubiquitylate substrates. How monoversus polyubiquitylation of substrates is mediated is unknown (1-4). Smurf1 and Smurf2 are HECT domain ubiquitin ligases that regulate transforming growth factor-␤ signaling as well as cell motility and polarity in part through targeting the GTPases RhoA and Rap1 as well as talin and core planar cell polarity components for polyubiquitin-dependent degradation (5-7).Recently, non-covalent ubiquitin binding to the HECT domain of Rsp5 was characterized and proposed to play a role in regulating polyubiquitylation (8). Here we employ NMR spectroscopy to map the non-covalent ubiquitin binding surface (UBS) 5 on the HECT domain of Smurf2. We show that mutation of a conserved surface tyrosine residue Tyr-459 on the UBS interferes with Smurf-dependent degradation of RhoA and blocks polyubiquitylation but not monoubiquitylation by the Smurf HECT domain. Furthermore, we show that efficient binding of a monoubiquitylated version of RhoA to the HECT domain is dependent on the UBS. Our results point to a model in which non-covalent binding of ubiquitin by HECT domains promotes polyubiquitylation by stabilizing interaction with monoubiquitylated substrates. EXPERIMENTAL PROCEDURESNMR Analysis-For NMR structure studies, ubiquitin (aa 1-76), the Smurf2 HECT domain (aa 366 -748), and its N2 (aa 519 -590) and C-lobe (aa 630 -748) subdomains were expressed in Escherichia coli BL21(DE3) CodonPlus cells upon induction with isopropyl 1-thio-␤-D-galactopyranoside. Cells...