Kenneth E. Prehoda

The PDZ protein interaction domain of neuronal nitric oxide synthase (nNOS) can heterodimerize with the PDZ domains of postsynaptic density protein 95 and syntrophin through interactions that are not mediated by recognition of a typical carboxyl-terminal motif. The nNOS-syntrophin PDZ complex structure revealed that the domains interact in an unusual linear head-to-tail arrangement. The nNOS PDZ domain has two opposite interaction surfaces-one face has the canonical peptide binding groove, whereas the other has a beta-hairpin "finger." This nNOS beta finger docks in the syntrophin peptide binding groove, mimicking a peptide ligand, except that a sharp beta turn replaces the normally required carboxyl terminus. This structure explains how PDZ domains can participate in diverse interaction modes to assemble protein networks.

The protein N-WASP [a homolog to the Wiskott-Aldrich syndrome protein (WASP)] regulates actin polymerization by stimulating the actin-nucleating activity of the actin-related protein 2/3 (Arp2/3) complex. N-WASP is tightly regulated by multiple signals: Only costimulation by Cdc42 and phosphatidylinositol (4,5)-bisphosphate (PIP2) yields potent polymerization. We found that regulation requires N-WASP's constitutively active output domain (VCA) and two regulatory domains: a Cdc42-binding domain and a previously undescribed PIP(2)-binding domain. In the absence of stimuli, the regulatory modules together hold the VCA-Arp2/3 complex in an inactive "closed" conformation. In this state, both the Cdc42- and PIP2-binding sites are masked. Binding of either input destabilizes the closed state and enhances binding of the other input. This cooperative activation mechanism shows how combinations of simple binding domains can be used to integrate and amplify coincident signals.

The active-site CXXC motif of thiol:disulfide oxidoreductases is essential for their catalysis of redox reactions. Changing the XX residues can perturb the reduction potential of the active-site disulfide bond of the Escherichia coli enzymes thioredoxin (Trx; CGPC) and DsbA (CPHC). The reduction potential is correlated with the acidity of the N-terminal cysteine residue of the CXXC motif. As the pKa is lowered, the disulfide bond becomes more easy to reduce. A change in pKa can account fully for a change in reduction potential in well-characterized CXXC motifs of DsbA but not of Trx. Formal analysis of the Nernst equation reveals that reduction potential contains both pH-dependent and pH-independent components. Indeed, the difference between the reduction potentials of wild-type Trx and wild-type DsbA cannot be explained solely by differences in thiol pKa values. Structural data for thiol:disulfide oxidoreductases reveal no single factor that determines the pH-independent component of the reduction potential. In addition, the pH-dependent component is complex when the redox state of the CXXC motif affects the titration of residues other than the thiols. These intricacies enable CXXC motifs to vary widely in their capacity to assist electron flow, and thereby engender a family of thiol:disulfide oxidoreductases that play diverse roles in biochemistry.