De novo mapping of α-helix recognition sites on protein surfaces using unbiased libraries.

The α-helix is one of the most common protein surface recognition motifs found in nature, and its unique amide-cloaking properties also enable α-helical polypeptide motifs to exist in membranes. Together, these properties have inspired the development of α-helically constrained (Helicon) therapeutics that can enter cells and bind targets that have been considered "undruggable", such as protein-protein interactions. To date, no general method for discovering α-helical binders to proteins has been reported, limiting Helicon drug discovery to only those proteins with previously characterized α-helix recognition sites, and restricting the starting chemical matter to those known α-helical binders. Here, we report a general and rapid screening method to empirically map the α-helix binding sites on a broad range of target proteins in parallel using large, unbiased Helicon phage display libraries and next-generation sequencing. We apply this method to screen six structurally diverse protein domains, only one of which had been previously reported to bind isolated α-helical peptides, discovering 20 families that collectively comprise several hundred individual Helicons. Analysis of 14 X-ray cocrystal structures reveals at least nine distinct α-helix recognition sites across these six proteins, and biochemical and biophysical studies show that these Helicons can block protein-protein interactions, inhibit enzymatic activity, induce conformational rearrangements, and cause protein dimerization. We anticipate that this method will prove broadly useful for the study of protein recognition and for the development of both biochemical tools and therapeutics for traditionally challenging protein targets.

Biochemical and structural characterization of a novel cooperative binding mode by Pit-1 with CATT repeats in the macrophage migration inhibitory factor promoter.

Overexpression of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) is linked to a number of autoimmune diseases and cancer. MIF production has been correlated to the number of CATT repeats in a microsatellite region upstream of the MIF gene. We have characterized the interaction of pituitary-specific positive transcription factor 1 (Pit-1) with a portion of the MIF promoter region flanking a microsatellite polymorphism (-794 CATT5-8). Using fluorescence anisotropy, we quantified tight complex formation between Pit-1 and an oligonucleotide consisting of eight consecutive CATT repeats (8xCATT) with an apparent Kd of 35 nM. Using competition experiments we found a 23 base pair oligonucleotide with 4xCATT repeats to be the minimum DNA sequence necessary for high affinity interaction with Pit-1. The stoichiometry of the Pit-1 DNA interaction was determined to be 2:1 and binding is cooperative in nature. We subsequently structurally characterized the complex and discovered a completely novel binding mode for Pit-1 in contrast to previously described Pit-1 complex structures. The affinity of Pit-1 for the CATT target sequence was found to be highly dependent on cooperativity. This work lays the groundwork for understanding transcriptional regulation of MIF and pursuing Pit-1 as a therapeutic target to treat MIF-mediated inflammatory disorders.

Structure and interactions of the C-terminal metal binding domain of Archaeoglobus fulgidus CopA.

The Cu(+)-ATPase CopA from Archaeoglobus fulgidus belongs to the P(1B) family of the P-type ATPases. These integral membrane proteins couple the energy of ATP hydrolysis to heavy metal ion translocation across membranes. A defining feature of P(1B-1)-type ATPases is the presence of soluble metal binding domains at the N-terminus (N-MBDs). The N-MBDs exhibit a conserved ferredoxin-like fold, similar to that of soluble copper chaperones, and bind metal ions via a conserved CXXC motif. The N-MBDs enable Cu(+) regulation of turnover rates apparently through Cu-sensitive interactions with catalytic domains. A. fulgidus CopA is unusual in that it contains both an N-terminal MBD and a C-terminal MBD (C-MBD). The functional role of the unique C-MBD has not been established. Here, we report the crystal structure of the apo, oxidized C-MBD to 2.0 A resolution. In the structure, two C-MBD monomers form a domain-swapped dimer, which has not been observed previously for similar domains. In addition, the interaction of the C-MBD with the other cytoplasmic domains of CopA, the ATP binding domain (ATPBD) and actuator domain (A-domain), has been investigated. Interestingly, the C-MBD interacts specifically with both of these domains, independent of the presence of Cu(+) or nucleotides. These data reinforce the uniqueness of the C-MBD and suggest a distinct structural role for the C-MBD in CopA transport.

Development of a structure-analysis pipeline using multiple-solvent crystal structures of barrier-to-autointegration factor.

The multiple-solvent crystal structure (MSCS) approach uses high concentrations of organic solvents to characterize the interactions and effects of solvents on proteins. Here, the method has been further developed and an MSCS data-handling pipeline is presented that uses the Detection of Related Solvent Positions (DRoP) program to improve data quality. DRoP is used to selectively model conserved water molecules, so that an advanced stage of structural refinement is reached quickly. This allows the placement of organic molecules more accurately and convergence on high-quality maps and structures. This pipeline was applied to the chromatin-associated protein barrier-to-autointegration factor (BAF), resulting in structural models with better than average statistics. DRoP and Phenix Structure Comparison were used to characterize the data sets and to identify a binding site that overlaps with the interaction site of BAF with emerin. The conserved water-mediated networks identified by DRoP suggested a mechanism by which water molecules are used to drive the binding of DNA. Normalized and differential B-factor analysis is shown to be a valuable tool to characterize the effects of specific solvents on defined regions of BAF. Specific solvents are identified that cause stabilization of functionally important regions of the protein. This work presents tools and a standardized approach for the analysis and comprehension of MSCS data sets.

Structure of the actuator domain from the Archaeoglobus fulgidus Cu(+)-ATPase.

Copper homeostasis is maintained in part by membrane-bound P(1B)-type ATPases that are found in all organisms and drive the transport of this essential, yet toxic, metal ion across cellular membranes. CopA from Archaeoglobus fulgidus is a hyperthermophilic member of this ATPase subfamily and is homologous to the human Wilson and Menkes disease ATPases. To gain insight into Cu(+)-ATPase function, the structure of the CopA actuator domain (A-domain) was determined to 1.65 A resolution. The CopA A-domain functions to couple ATP hydrolysis in the ATP binding domain (ATPBD) with structural rearrangements of critical transmembrane segments. Its fold is quite similar to that of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1) A-domain, with the exception of an external loop region. On the basis of sequence and structural comparisons, specific residues that probably interact with the CopA ATPBD have been identified. Comparisons to the Wilson and Menkes disease A-domains reveal the presence of an additional loop that may be associated with regulatory functions in eukaryotic Cu(+)-ATPases. Finally, several mutations in the Wilson and Menkes disease ATPases occur in the A-domain, and their likely effects on function can be inferred from the CopA A-domain structure.