Molecular recognition of protein surfaces: high affinity ligands for the CBP KIX domain

Potent and specific inhibitors of protein.protein interactions have potential both as therapeutic compounds and biological tools, yet discovery of such molecules remains a challenge. Our laboratory has recently described a strategy, called protein grafting, for the identification of miniature proteins that bind protein surfaces with high affinity and specificity and inhibit the formation of protein.protein complexes. In protein grafting, those residues that comprise a functional alpha-helical binding epitope are stabilized on the solvent-exposed alpha-helical face of the small yet stable protein avian pancreatic polypeptide (aPP). Here we use protein grafting in combination with molecular evolution by phage display to identify phosphorylated peptide ligands that recognize the shallow surface of CBP KIX with high nanomolar to low micromolar affinity. Furthermore, we show that grafting of the CBP KIX-binding epitope of CREB KID onto the aPP scaffold yields molecules capable of high affinity recognition of CBP KIX even in the absence of phosphorylation. Importantly, both classes of designed ligands exhibit high specificity for the target CBP KIX domain over carbonic anhydrase and calmodulin, two unrelated proteins that bind hydrophobic or alpha-helical molecules that might be encountered in vivo.     

Discovery and characterization of a cell-permeable, small-molecule c-Abl kinase activator that binds to the myristoyl binding site

c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the αI helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the αI helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.     

Helical Propensity in an Intrinsically Disordered Protein Accelerates Ligand Binding

Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well‐ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activation domain of the activator for thyroid hormone and retinoid receptors (ACTR) is intrinsically disordered and folds upon binding to the nuclear coactivator binding domain (NCBD) of the CREB binding protein. A number of mutants was designed that selectively perturbs the amount of secondary structure in unbound ACTR without interfering with the intermolecular interactions between ACTR and NCBD. Using NMR spectroscopy and fluorescence‐monitored stopped‐flow kinetic measurements we show that the secondary structure content in helix 1 of ACTR indeed influences the binding kinetics. The results thus support the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins.     

Partial high-resolution structure of phosphorylated and non-phosphorylated leucine-rich amelogenin protein adsorbed to hydroxyapatite

The formation of biogenic materials requires the interaction of organic molecules with the mineral phase. In forming enamel, the amelogenin proteins contribute to the mineralization of hydroxyapatite (HAp). Leucine-rich amelogenin protein (LRAP) is a naturally occurring splice variant of amelogenin that comprises amelogenin's predicted HAp binding domains. We determined the partial structure of phosphorylated and non-phosphorylated LRAP variants bound to HAp using combined solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction. New ssNMR measurements in the N-terminus indicate a largely extended structure for both variants, though some measurements are consistent with a partially helical N-terminal segment. The N-terminus of the phosphorylated variant is found to be consistently closer to the HAp surface than the non-phosphorylated variant. Structure prediction was biased using 21 ssNMR measurements in the N- and C-terminus at five HAp crystal faces. The predicted fold of LRAP is similar at all HAp faces studied, regardless of phosphorylation. Largely consistent with experimental observations, LRAP's predicted structure is relatively extended with a helix-turn-helix motif in the N-terminal domain and some helix in the C-terminal domain, and the N-terminal domain of the phosphorylated variant binds HAp more closely than the N-terminal domain of the non-phosphorylated variant. Predictions for both variants show some potential binding specificity for the {010} HAp crystal face, providing further support that amelogenins block crystal growth on the a and b faces to allow elongated crystals in the c-axis.     

Beyond the BET Family: Targeting CBP/p300 with 4‐Acyl Pyrroles

Bromodomain and extra‐terminal domain (BET) inhibitors are widely used both as chemical tools to study the biological role of their targets in living organisms and as candidates for drug development against several cancer variants and human disorders. However, non‐BET bromodomains such as those in p300 and CBP are less studied. XDM‐CBP is a highly potent and selective inhibitor for the bromodomains of CBP and p300 derived from a pan‐selective BET BRD‐binding fragment. Along with X‐ray crystal‐structure analysis and thermodynamic profiling, XDM‐CBP was used in screenings of several cancer cell lines in vitro to study its inhibitory potential on cancer cell proliferation. XDM‐CBP is demonstrated to be a potent and selective CBP/p300 inhibitor that acts on specific cancer cell lines, in particular malignant melanoma, breast cancer, and leukemia.     

A free-energy landscape for coupled folding and binding of an intrinsically disordered protein in explicit solvent from detailed all-atom computations

The N-terminal repressor domain of neural restrictive silencer factor (NRSF) is an intrinsically disordered protein (IDP) that binds to the paired amphipathic helix (PAH) domain of mSin3. An NMR experiment revealed that the minimal binding unit of NRSF is a 15-residue segment that adopts a helical structure upon binding to a cleft of mSin3. We computed a free-energy landscape of this system by an enhanced conformational sampling method, all-atom multicanonical molecular dynamics. The simulation started from a configuration where the NRSF segment was fully disordered and distant from mSin3 in explicit solvent. In the absence of mSin3, the disordered NRSF segment thermally fluctuated between hairpins, helices, and bent structures. In the presence of mSin3, the segment bound to mSin3 by adopting the structures involved in the isolated state, and non-native and native complexes were formed. The free-energy landscape comprised three superclusters, and free-energy barriers separated the superclusters. The native complex was located at the center of the lowest free-energy cluster. When NRSF landed in the largest supercluster, the generated non-native complex moved on the landscape to fold into the native complex, by increasing the interfacial hydrophobic contacts and the helix content. When NRSF landed in other superclusters, the non-native complex overcame the free-energy barriers between the various segment orientations in the binding cleft of mSin3. Both population-shift and induced-fit (or induced-folding) mechanisms work cooperatively in the coupled folding and binding. The diverse structural adaptability of NRSF may be related to the hub properties of the IDP.     

The Mechanism of p53 Rescue by SUSP4

p53 is an important tumor‐suppressor protein deactivation of which by mdm2 results in cancers. A SUMO‐specific protease 4 (SUSP4) was shown to rescue p53 from mdm2‐mediated deactivation, but the mechanism is unknown. The discovery by NMR spectroscopy of a “p53 rescue motif” in SUSP4 that disrupts p53‐mdm2 binding is presented. This 29‐residue motif is pre‐populated with two transient helices connected by a hydrophobic linker. The helix at the C‐terminus binds to the well‐known p53‐binding pocket in mdm2 whereas the N‐terminal helix serves as an affinity enhancer. The hydrophobic linker binds to a previously unidentified hydrophobic crevice in mdm2. Overall, SUSP4 appears to use two synergizing modules, the p53 rescue motif described here and a globular‐structured SUMO‐binding catalytic domain, to stabilize p53. A p53 rescue motif peptide exhibits an anti‐tumor activity in cancer cell lines expressing wild‐type p53. A pre‐structures motif in the intrinsically disordered proteins is thus important for target recognition.     

The Molecular Mechanism of P2Y1 Receptor Activation

Human purinergic G protein‐coupled receptor P2Y₁ (P2Y₁R) is activated by adenosine 5′‐diphosphate (ADP) to induce platelet activation and thereby serves as an important antithrombotic drug target. Crystal structures of P2Y₁R revealed that one ligand (MRS2500) binds to the extracellular vestibule of this GPCR, whereas another (BPTU) occupies the surface between transmembrane (TM) helices TM2 and TM3. We introduced a total of 20 μs all‐atom long‐timescale molecular dynamic (MD) simulations to inquire why two molecules in completely different locations both serve as antagonists while ADP activates the receptor. Our results indicate that BPTU acts as an antagonist by stabilizing extracellular helix bundles leading to an increase of the lipid order, whereas MRS2500 blocks signaling by occupying the ligand binding site. Both antagonists stabilize an ionic lock within the receptor. However, binding of ADP breaks this ionic lock, forming a continuous water channel that leads to P2Y₁R activation.