P. Andrew Chong

Coupling of tandem Smad ubiquitination regulatory factor (Smurf) WW domains modulates target specificity

Smad ubiquitination regulatory factor 2 (Smurf2) is an E3 ubiquitin ligase that participates in degradation of TGF-β receptors and other targets. Smurf2 WW domains recognize PPXY (PY) motifs on ubiquitin ligase target proteins or on adapters, such as Smad7, that bind to E3 target proteins. We previously demonstrated that the isolated WW3 domain of Smurf2, but not the WW2 domain, can directly bind to a Smad7 PY motif. We show here that the WW2 augments this interaction by binding to the WW3 and making auxiliary contacts with the PY motif and a novel E/D-S/T-P motif, which is N-terminal to all Smad PY motifs. The WW2 likely enhances the selectivity of Smurf2 for the Smad proteins. NMR titrations confirm that Smad1 and Smad2 are bound by Smurf2 with the same coupled WW domain arrangement used to bind Smad7. The analogous WW domains in the short isoform of Smurf1 recognize the Smad7 PY peptide using the same coupled mechanism. However, a longer Smurf1 isoform, which has an additional 26 residues in the inter-WW domain linker, is only partially able to use the coupled WW domain binding mechanism. The longer linker results in a decrease in affinity for the Smad7 peptide. Interdomain coupling of WW domains enhances selectivity and enables the tuning of interactions by isoform switching.

An Expanded WW Domain Recognition Motif Revealed by the Interaction between Smad7 and the E3 Ubiquitin Ligase Smurf2.

Smurf2 is an E3 ubiquitin ligase that drives degradation of the transforming growth factor-β receptors and other targets. Recognition of the receptors by Smurf2 is accomplished through an intermediary protein, Smad7. Here we have demonstrated that the WW3 domain of Smurf2 can directly bind to the Smad7 polyproline-tyrosine (PY) motif. Of particular interest, the highly conserved WW domain binding site Trp, which interacts with target PY motifs, is a Phe in the Smurf2 WW3 domain. To examine this interaction, the solution structure of the complex between the Smad7 PY motif region (ELESPPPPYSRYPMD) and the Smurf2 WW3 domain was determined. The structure reveals that, in addition to binding the PY motif, the WW3 domain binds six residues C-terminal to the PY motif (PY-tail). Although the Phe in the WW3 domain binding site decreases affinity relative to the canonical Trp, this is balanced by additional interactions between the PY-tail and the β1-strand and β1–β2 loop of the WW3 domain. The interaction between the Smurf2 WW3 domain and the Smad7 PY motif is the first example of PY motif recognition by a WW domain with a Phe substituted for the binding site Trp. This unusual interaction allows the Smurf2 WW3 domain to recognize a subset of PY motif-containing proteins utilizing an expanded surface to provide specificity.

Structural changes of CFTR R region upon phosphorylation: a plastic platform for intramolecular and intermolecular interactions

Chloride channel gating and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) are regulated by phosphorylation. Intrinsically disordered segments of the protein are responsible for phospho‐regulation, particularly the regulatory (R) region that is a target for several kinases and phosphatases. The R region remains disordered following phosphorylation, with different phosphorylation states sampling various conformations. Recent studies have demonstrated the crucial role that intramolecular and intermolecular interactions of the R region play in CFTR regulation. Different partners compete for the same binding segment, with the R region containing multiple overlapping binding elements. The non‐phosphorylated R region interacts with the nucleotide binding domains and inhibits channel activity by blocking heterodimerization. Phosphorylation shifts the equilibrium such that the R region is excluded from the dimer interface, facilitating gating and processing by stimulating R region interactions with other domains and proteins. The dynamic conformational sampling and transient binding of the R region to multiple partners enables complex control of CFTR channel activity and trafficking.

Conformational Changes Relevant to Channel Activity and Folding within the first Nucleotide Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator

Background: The CFTR chloride channel undergoes conformational changes during its gating cycle. Results: H620Q mutation associated with increased channel Po, and the corrector/potentiator CFFT-001 both lead to similar conformational shifts in NBD1. Conclusion: There is an intrinsic conformational equilibrium within NBD1 that is correlated with channel activity. Significance: Conformational fluctuations within NBD1 are fundamental to CFTR regulation. Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between β-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with β-strands S3/S9/S10, consistent with docking simulations. Decreases in Tm from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics.

Liquid–liquid phase separation in cellular signaling systems

Liquid-liquid demixing or phase separation of protein with RNA is now recognized to be a key part of the mechanism for assembly of ribonucleoprotein granules. Cellular signaling also appears to employ phase separation as a mechanism for amplification or control of signal transduction both within the cytoplasm and at the membrane. The concept of receptor clustering, identified more than 3 decades ago, is now being examined through the lens of phase separation leading to new insights. Intrinsically disordered proteins or regions are central to these processes owing to their flexibility and accessibility for dynamic protein-protein interactions and post-translational modifications. We review some recent examples, examine the mechanisms driving phase separation and delineate the implications for signal transduction systems.

Structural, Functional, and Bioinformatic Studies Demonstrate the Crucial Role of an Extended Peptide Binding Site for the SH3 Domain of Yeast Abp1p

SH3 domains, which are among the most frequently occurring protein interaction modules in nature, bind to peptide targets ranging in length from 7 to more than 25 residues. Although the bulk of studies on the peptide binding properties of SH3 domains have focused on interactions with relatively short peptides (less than 10 residues), a number of domains have been recently shown to require much longer sequences for optimal binding affinity. To gain greater insight into the binding mechanism and biological importance of interactions between an SH3 domain and extended peptide sequences, we have investigated interactions of the yeast Abp1p SH3 domain (AbpSH3) with several physiologically relevant 17-residue target peptide sequences. To obtain a molecular model for AbpSH3 interactions, we solved the structure of the AbpSH3 bound to a target peptide from the yeast actin patch kinase, Ark1p. Peptide target complexes from binding partners Scp1p and Sjl2p were also characterized, revealing that the AbpSH3 uses a common extended interface for interaction with these peptides, despite Kd values for these peptides ranging from 0.3 to 6 μm. Mutagenesis studies demonstrated that residues across the whole 17-residue binding site are important both for maximal in vitro binding affinity and for in vivo function. Sequence conservation analysis revealed that both the AbpSH3 and its extended target sequences are highly conserved across diverse fungal species as well as higher eukaryotes. Our data imply that the AbpSH3 must bind extended target sites to function efficiently inside the cell.

Dynamics Intrinsic to Cystic Fibrosis Transmembrane Conductance Regulator Function and Stability

The cystic fibrosis transmembrane conductance regulator (CFTR) requires dynamic fluctuations between states in its gating cycle for proper channel function, including changes in the interactions between the nucleotide-binding domains (NBDs) and between the intracellular domain (ICD) coupling helices and NBDs. Such motions are also linked with fluctuating phosphorylation-dependent binding of CFTRs disordered regulatory (R) region to the NBDs and partners. Folding of CFTR is highly inefficient, with the marginally stable NBD1 sampling excited states or folding intermediates that are aggregation-prone. The severe CF-causing F508del mutation exacerbates the folding inefficiency of CFTR and leads to impaired channel regulation and function, partly as a result of perturbed NBD1-ICD interactions and enhanced sampling of these NBD1 excited states. Increased knowledge of the dynamics within CFTR will expand our understanding of the regulated channel gating of the protein as well as of the F508del defects in folding and function.

Direct Binding of the Corrector VX-809 to Human CFTR NBD1: Evidence of an Allosteric Coupling between the Binding Site and the NBD1:CL4 Interface

Understanding the mechanism of action of modulator compounds for the cystic fibrosis transmembrane conductance regulator (CFTR) is key for the optimization of therapeutics as well as obtaining insights into the molecular mechanisms of CFTR function. We demonstrate the direct binding of VX-809 to the first nucleotide-binding domain (NBD1) of human CFTR. Disruption of the interaction between C-terminal helices and the NBD1 core upon VX-809 binding is observed from chemical shift changes in the NMR spectra of residues in the helices and on the surface of β-strands S3, S9, and S10. Binding to VX-809 leads to a significant negative shift in NBD1 thermal melting temperature (Tm), pointing to direct VX-809 interaction shifting the NBD1 conformational equilibrium. An inter-residue correlation analysis of the chemical shift changes provides evidence of allosteric coupling between the direct binding site and the NBD1:CL4 interface, thus enabling effects on the interface in the absence of direct binding in that location. These NMR binding data and the negative Tm shifts are very similar to those previously reported by us for binding of the dual corrector-potentiator CFFT-001 to NBD1 (Hudson et al., 2012), suggesting that the two compounds may share some aspects of their mechanisms of action. Although previous studies have shown an important role for VX-809 in modulating the conformation of the first membrane spanning domain (Aleksandrov et al., 2012; Ren et al., 2013), this additional mode of VX-809 binding provides insight into conformational dynamics and allostery within CFTR.

Binding screen for cftr correctors finds new chemical matter and yields insights into cf therapeutic strategy

The most common mutation in cystic fibrosis (CF) patients is deletion of F508 (ΔF508) in the first nucleotide binding domain (NBD1) of the CF transmembrane conductance regulator (CFTR). ΔF508 causes a decrease in the trafficking of CFTR to the cell surface and reduces the thermal stability of isolated NBD1; it is well established that both of these effects can be rescued by additional revertant mutations in NBD1. The current paradigm in CF small molecule drug discovery is that, like revertant mutations, a path may exist to ΔF508 CFTR correction through a small molecule chaperone binding to NBD1. We, therefore, set out to find small molecule binders of NBD1 and test whether it is possible to develop these molecules into potent binders that increase CFTR trafficking in CF‐patient‐derived human bronchial epithelial cells. Several fragments were identified that bind NBD1 at either the CFFT‐001 site or the BIA site. However, repeated attempts to improve the affinity of these fragments resulted in only modest gains. Although these results cannot prove that there is no possibility of finding a high‐affinity small molecule binder of NBD1, they are discouraging and lead us to hypothesize that the nature of these two binding sites, and isolated NBD1 itself, may not contain the features needed to build high‐affinity interactions. Future work in this area may, therefore, require constructs including other domains of CFTR in addition to NBD1, if high‐affinity small molecule binding is to be achieved.

Binding screen for cystic fibrosis transmembrane conductance regulator correctors finds new chemical matter and yields insights into cystic fibrosis therapeutic strategy

The most common mutation in cystic fibrosis (CF) patients is deletion of F508 (ΔF508) in the first nucleotide binding domain (NBD1) of the CF transmembrane conductance regulator (CFTR). ΔF508 causes a decrease in the trafficking of CFTR to the cell surface and reduces the thermal stability of isolated NBD1; it is well established that both of these effects can be rescued by additional revertant mutations in NBD1. The current paradigm in CF small molecule drug discovery is that, like revertant mutations, a path may exist to ΔF508 CFTR correction through a small molecule chaperone binding to NBD1. We, therefore, set out to find small molecule binders of NBD1 and test whether it is possible to develop these molecules into potent binders that increase CFTR trafficking in CF‐patient‐derived human bronchial epithelial cells. Several fragments were identified that bind NBD1 at either the CFFT‐001 site or the BIA site. However, repeated attempts to improve the affinity of these fragments resulted in only modest gains. Although these results cannot prove that there is no possibility of finding a high‐affinity small molecule binder of NBD1, they are discouraging and lead us to hypothesize that the nature of these two binding sites, and isolated NBD1 itself, may not contain the features needed to build high‐affinity interactions. Future work in this area may, therefore, require constructs including other domains of CFTR in addition to NBD1, if high‐affinity small molecule binding is to be achieved.