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...

The Par-1/MARK protein kinases play a pivotal role in establishing cellular polarity. This family of kinases contains a unique domain architecture, in which a ubiquitin-associated (UBA) domain is located C-terminal to the kinase domain. We have used a combination of x-ray crystallography and NMR dynamics experiments to understand the interaction of the human (h) MARK3 UBA domain with the adjacent kinase domain as compared with ubiquitin. The x-ray crystal structure of the linked hMARK3 kinase and UBA domains establishes that the UBA domain forms a stable intramolecular interaction with the N-terminal lobe of the kinase domain. However, solution-state NMR studies of the isolated UBA domain indicate that it is highly dynamic, undergoing conformational transitions that can be explained by a folding-unfolding equilibrium. NMR titration experiments indicated that the hMARK3 UBA domain has a detectable but extremely weak affinity for monoubiquitin, which suggests that conformational instability of the isolated hMARK3 UBA domain attenuates binding to ubiquitin despite the presence of residues typically involved in ubiquitin recognition. Our data identify a molecular mechanism through which the hMARK3 UBA domain has evolved to bind the kinase domain, in a fashion that stabilizes an open conformation of the Nand C-terminal lobes, at the expense of its capacity to engage ubiquitin. These results may be relevant more generally to the 30% of UBA domains that lack significant ubiquitin-binding activity, and they suggest a unique mechanism by which interaction domains may evolve new binding properties.catalytic domain ͉ dynamics ͉ Par-1 ͉ relaxation ͉ ubiquitin-associated

SummaryRNase G is the endoribonuclease responsible for forming the mature 5 ¢ ¢ ¢ ¢ end of 16S rRNA. This enzyme shares 35% identity with and 50% similarity to the Nterminal 470 amino acids encompassing the catalytic domain of RNase E, the major endonuclease in Escherichia coli . In this study, we developed nondenaturing purifications for overexpressed RNase G. Using mass spectrometry and N-terminal sequencing, we unambiguously identified the N-terminal sequence of the protein and found that translation is initiated at the second of two potential start sites. Using velocity sedimentation and oxidative cross-linking, we determined that RNase G exists largely as a dimer in equilibrium with monomers and higher multimers. Moreover, dimerization is required for activity. Four of the six cysteine residues of RNase G were mutated to serine. No single cysteine to serine mutation resulted in a complete loss of cross-linking, dimerization or activity. However, multiple mutations in a highly conserved cluster of cysteines, including C405 and C408, resulted in a partial loss of activity and a shift in the distribution of RNase G multimers towards monomers. We propose that many of the cysteines in RNase G lie on its surface and define, in part, the subunitsubunit interface.

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Understanding protein-protein interactions is a key step in unravelling the roles proteins play in cellular function. The ability to analyse protein-protein interactions rapidly and economically is a powerful research tool. Using peptide SPOT arrays, peptides of known sequence can be synthesized directly in discrete spots on a cellulose membrane and assayed for an interaction with a protein of interest. Several hundred peptides can be synthesized on each cellulose membrane; therefore, this method is amenable to designing high-throughput peptide binding studies. SPOT arrays are particularly well suited for deducing peptidic binding motifs within proteins that are difficult to purify in sufficient quantities for traditional biochemical analyses, as well as for determining binding specificities and targets for proteins of undefined function. Peptide SPOT arrays have been used extensively to define protein-protein interaction surfaces. In this chapter, we will outline the steps involved in designing and probing a peptide SPOT array to identify peptide binding motifs for a protein of interest.

We have examined the effect of herbimycin A, a benzoquinoid ansamycin antibiotic, on the pattern of gene expression in amphibians. Exposure of Xenopus laevis A6 kidney epithelial cells to 1 microgram/mL herbimycin A induced the synthesis of the heat shock proteins hsp30 and hsp70 as well as 33- and 45-kDa proteins. Enhanced synthesis of a 34-kDa protein appears to be specific to herbimycin A because its synthesis did not increase after heat shock (35 degrees C). In addition, the synthesis of hsp30 and hsp70 induced by herbimycin A was accompanied by an increase in their mRNAs. Herbimycin A induced a transient accumulation of hsp30 and hsp70 mRNA, which peaked between 4 and 6 h. Finally, concurrent treatment of cells with 0.5 microgram/mL herbimycin A and a mild heat shock of 27 degrees C yielded a synergistic accumulation of hsp30 and hsp70 mRNA. These studies demonstrate that herbimycin A can induce the expression of a set of stress proteins in amphibians and that concurrent treatment with herbimycin A and mild heat shock has a synergistic effect on the accumulation of hsp30 and hsp70 mRNA.