Our data show that helix H4 of the UBA domain of Mex67 influences its interaction with other partners in different ways. Thus, whereas in vitro deletion of helix H4 or the F596A mutation increases the affinity of the UBA domain for ubiquitin and ubiquitin multimers, it reduces the affinity for Hpr1. Moreover, deletion of helix H4 reduces both cotranscriptional recruitment of Mex67 to mRNPs and generates an mRNA export defect at 37°C.
It is unlikely that deletion of helix H4 results in a complete loss of structure of the UBA domain, because these constructs retain the ability to bind ubiquitin. Instead, helix H4 seems to be involved directly in Hpr1 binding, and the pronounced mRNA export defect seen in mex67
cells would be consistent with the Hpr1:Mex67UBA interaction being an important step in the overall export pathway. However, it seems unlikely that helix H4 alone can account for all of the effects seen with deletion of the entire UBA domain, because previous studies have, for example, shown a role for the intact domain in interacting with phenylalanine-glycine nucleoporins to facilitate translocation through nuclear pore complexes (Grant et al., 2003
). Our results indicate that the core of the UBA domain (helices H1–H3) is responsible for ubiquitin binding, whereas H4 participates to Hpr1 binding and also seems to regulate the interaction of the Mex67 UBA domain with ubiquitin. It should be noted that although H4 is necessary for Hpr1 binding, it is likely not sufficient for this interaction as suggested by the residues within the UBA domain that are affected upon Hpr1 binding in NMR spectra (our unpublished observations). Because both ubiquitin binding and Hpr1 stabilization are important for cotranscriptional recruitment and mRNA export, interfering with either UBA domain or H4 results in both cotranscriptional recruitment and mRNA export defects.
Based on these results, we hypothesize that, in vivo, helix H4 may function to coordinate the ubiquitin binding activity of the Mex67 UBA domain with recognition of specific substrates, in particular Hpr1, and also prevent binding to nonspecific substrates. Such a function would probably require a two-stage mechanism for the recognition of ubiquitinated Hpr1 by UBA-Mex67, with an initial helix H4-dependent binding of Hpr1 followed by an interaction with ubiquitins conjugated to Hpr1. This type of UBA-Mex67:ubiquitin interaction might also occur with other partners. Substrate specificity and ubiquitin linkage selectivity of UBA domains seem to be interdependent and do not exclusively rely on intrinsic properties of UBA domains, but rather they are precisely controlled by their molecular environment, as we show here for UBA-Mex67. For example, a specific interaction of the HIV-1 accessory protein Vpr with hHR23A-UBA1 domain involves loop 1 (Withers-Ward et al., 2000
), whereas interaction of the N-terminal Ubiquitin-like domain of hHR23A with hHR23A-UBA1 modulates its selectivity for polyubiquitin linkage.
Comparison of the structure of the Mex67 UBA domain with the structures of other UBA:ubiquitin conjugates indicates that much of the interaction surface has been retained. For example, when the structure of the Mex67 UBA domain is superimposed on the UBA domain in the Ede1:ubiquitin complex (Swanson et al., 2006
), much of the general character and structure of the interaction interface is retained for helix H3 (). How might helix H4 influence the interactions? Steric hindrance seems unlikely, because in our model for the UBA-Mex67:monoubiquitin complex, helix H4 does not contact ubiquitin, although steric hindrance may possibly be important for binding of Lys48-linked ubiquitin multimers. Alternatively, previous studies have shown that the structure of the TAP-UBA domain is relatively plastic and mutations can introduce movements of helices H1, H2, and H3 that significantly alter interactions with other partners. Structural studies revealed that helix H4 in TAP-UBA is highly mobile and undergoes major conformational changes upon binding FXFG repeats or in the F617A mutant (Grant et al., 2003
). This result, together with the involvement of helix H4 in the interaction with Hpr1, would be consistent with the conformational change observed by SPR being associated with a change in helix H4 being triggered by binding to Hpr1. However, a change in the overall structure of the UBA domain associated with its binding Hpr1cannot be formally excluded. In either case, the conformational change induced by Hpr1 would facilitate ubiquitin binding by the UBA domain.
Although further work will be required to define the precise manner in which helix H4 modulates the interactions of the Mex67 UBA domain, our present studies demonstrate how the binding of Hpr1 can influence the affinity of this domain for ubiquitin and thereby provide a novel mechanism by which interactions between different components of the mRNP processing and export machinery can be modulated.