Abstract
Guanine nucleotide exchange factors (GEFs), which activate small GTP-binding proteins (SMG) by stimulating their GDP/GTP exchange, are emerging as candidate targets for the inhibition of cellular pathways involved in diseases. However, their specific inhibition by competitive inhibitors is challenging, because GEF and SMG families comprise highly similar members. Nature shows us an alternative strategy called interfacial inhibition, exemplified by Brefeldin A (BFA). BFA inhibits the activation of Arf1 by its GEFs in vivo by stabilizing an abortive complex between Arf-GDP and the catalytic Sec7 domain of some of its GEFs. Here we characterize the specificity of BFA toward wild-type (ARNO and BIG1) and mutant Sec7 domains and toward class I, II, and III Arfs. We find that BFA sensitivity of the exchange reaction depends on the nature of both the Sec7 domain and the Arf protein. A single Phe/Tyr substitution is sufficient to achieve BFA sensitivity of the Sec7 domain, which is supported by our characterization of brefeldin C (BFC), a BFA analog that cannot interact with the Tyr residue, and by free energy computations. We further show that Arf1 and Arf5, but not Arf6, are BFA-sensitive, despite their having every BFA-interacting residue in common. Analysis of Arf6 mutants points to the dynamics of the interswitch, which is involved in membrane-to-nucleotide signal propagation, as contributing to, although not sufficient for, BFA sensitivity. Altogether, our results reveal the Tyr/Phe substitution as a novel tool for monitoring BFA sensitivity of cellular ArfGEFs and document the exquisite and dual specificity that can be achieved by an interfacial inhibitor.
Highlights
Guanine nucleotide exchange factors (GEFs),7 which activate small GTP-binding proteins (SMGs) by stimulating their GDP/GTP exchange, are increasingly being characterized as critical determinants in SMG signaling specificity
Brefeldin A (BFA)-sensitizing Mutations in ARNO Have No Impact on the Specific Exchange Activity—In order to analyze the specificity of BFA in terms of both the sequence of the Sec7 domain and the class of its Arf substrate, we first characterized a model system in which these parameters can be varied
We characterized the respective contributions of Arf proteins from classes I, II, and III and from the sequence of the Sec7 domain to conferring sensitivity of the exchange reaction to interfacial inhibition by BFA or a BFA analog
Summary
Protein Expression, and Purification—Point mutations were introduced using the QuikChangeTM site-directed mutagenesis kit (Stratagene, Amsterdam, The Netherlands). ARNO constructs were expressed and purified essentially as described in Ref. 37 except that cells were lysed by sonication, and protein purity was polished, after the ion exchange step on a Q-Sepharose fast flow column (Amersham Biosciences), by gel filtration on a Superdex 75 XK 16/90 column (Amersham Biosciences) equilibrated with a 50 mM TrisHCl, pH 8, buffer containing 1 mM EDTA and 0.5 mM -mercaptoethanol. Protein solutions were incubated 5 min at 37 °C in 700 l of reaction buffer (50 mM Tris-HCl, pH 8, 50 mM NaCl, 2 mM MgCl2, 2 mM -mercaptoethanol) with or without BFA or BFC. Molecular Dynamics Simulations and Free Energy Analysis—Molecular dynamics (MD) used the Charmm force field [43] for the proteins, Mg2ϩ, and GDP. Complex np was modeled as proportional to the loss in solventaccessible surface area, which was calculated with CHARMM using a probe radius of 1.4 Å and a ␥ constant of 5 cal1⁄7molϪ11⁄7ÅϪ1 according to the equation,
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