The cytidine repressor (CytR) is a member of the LacR family of bacterial repressors with distinct functional features. The Escherichia coli CytR regulon comprises nine operons whose palindromic operators vary in both sequence and, most significantly, spacing between the recognition half-sites. This suggests a strong likelihood that protein folding would be coupled to DNA binding as a mechanism to accommodate the variety of different operator architectures to which CytR is targeted. Such coupling is a common feature of sequence-specific DNA-binding proteins, including the LacR family repressors; however, there are no significant structural rearrangements upon DNA binding within the three-helix DNA-binding domains (DBDs) studied to date. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the CytR DBD free in solution and to determine the high-resolution structure of a CytR DBD monomer bound specifically to one DNA half-site of the uridine phosphorylase (udp) operator. We find that the free DBD populates multiple distinct conformations distinguished by up to four sets of NMR peaks per residue. This structural heterogeneity is previously unknown in the LacR family. These stable structures coalesce into a single, more stable udp-bound form that features a three-helix bundle containing a canonical helix-turn-helix motif. However, this structure differs from all other LacR family members whose structures are known with regard to the packing of the helices and consequently their relative orientations. Aspects of CytR activity are unique among repressors; we identify here structural properties that are also distinct and that might underlie the different functional properties.

Aromatic polyketides are an important class of natural products that possess a wide range of biological activities. The cyclization of the polyketide chain is a critical control point in the biosynthesis of aromatic polyketides. The aromatase/cyclases (ARO/CYCs) are an important component of the Type II polyketide synthase (PKS) and help fold the polyketide for regiospecific cyclizations of the first ring and/or aromatization, promoting two commonly observed first-ring cyclization patterns for the bacterial Type II PKSs: C7–C12 and C9–C14. We had previously reported the crystal structure and enzymological analyses of the TcmN ARO/CYC, which promotes C9–C14 first-ring cyclization. However, how C7–C12 first-ring cyclization is controlled remains unresolved. In this work, we present the 2.4 Å crystal structure of ZhuI, a C7–C12-specific first-ring ARO/CYC from the Type II PKS pathway responsible for the production of the R1128 polyketides. Though ZhuI possesses a helix-grip fold shared by TcmN ARO/CYC, there are substantial differences in overall structure and pocket residue composition to implicate the preference for directing C7–C12 (rather than C9–C14) cyclization. Docking studies and site-directed mutagenesis coupled to an in vitro activity assay demonstrate that ZhuI pocket residues R66, H109, and D146 are important for enzyme function. The ZhuI crystal structure helps visualize the structure and putative dehydratase function of the di-domain ARO/CYCs from KR-containing Type II PKSs. The sequence-structure-function analysis described for ZhuI elucidates the molecular mechanisms that control C7–C12 first-ring polyketide cyclization and builds a foundation for future endeavors into directing cyclization patterns for engineered biosynthesis of aromatic polyketides.

GTPases and kinases are two predominant signaling modules that regulate cell fate. Dysregulation of Ras, a GTPase, and the three eponymous kinases that form key nodes of the associated phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/AKT/mTOR pathway have been implicated in many cancers, including pancreatic cancer, a disease noted for its current lack of effective therapeutics. The K-Ras isoform of Ras is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC) and there is growing evidence linking aberrant PI3K/AKT/mTOR pathway activity to PDAC. Although these observations suggest that targeting one of these nodes might lead to more effective treatment options for patients with pancreatic and other cancers, the complex regulatory mechanisms and the number of sequence-conserved isoforms of these proteins have been viewed as significant barriers in drug development. Emerging insights into the allosteric regulatory mechanisms of these proteins suggest novel opportunities for development of selective allosteric inhibitors with fragment-based drug discovery (FBDD) helping make significant inroads. The fact that allosteric inhibitors of Ras and AKT are currently in pre-clinical development lends support to this approach. In this article, we will focus on the recent advances and merits of developing allosteric drugs targeting these two inter-related signaling pathways.

The E. coli cytidine repressor (CytR) is a member of the LacR family of bacterial repressors that regulates nine operons with distinct spacing and orientations of recognition sites. Understanding the structural features of the CytR DNA-binding domain (DBD) when bound to DNA is critical to understanding differential mechanisms of gene regulation. We previously reported the structure of the CytR DBD monomer bound specifically to half-site DNA and found that the DBD exists as a three-helix bundle containing a canonical helix-turn-helix motif, similar to other proteins that interact with DNA [Moody, et al (2011), Biochemistry 50:6622-32]. We also studied the free state of the monomer and found that since NMR spectra show it populates up to four distinct conformations, the free state exists as an intrinsically disordered protein (IDP). Here, we present further analysis of the DBD structure and dynamics in the context of full-site operator or nonspecific DNA. DBDs bound to full-site DNA show one set of NMR signals, consistent with fast exchange between the two binding sites. When bound to full-length DNA, we observed only slight changes in structure compared to the monomer structure and no folding of the hinge helix. Notably, the CytR DBD behaves quite differently when bound to nonspecific DNA compared to LacR. A dearth of NOEs and complete lack of protection from hydrogen exchange are consistent with the protein populating a flexible, molten state when associated with DNA nonspecifically, similar to fuzzy complexes. The CytR DBD structure is significantly more stable when bound specifically to the udp half-site substrate. For CytR, the transition from nonspecific association to specific recognition results in substantial changes in protein mobility that are coupled to structural rearrangements. These effects are more pronounced in the CytR DBD compared to other LacR family members.

Dystrophin is a 427 kDa rod-shaped protein encoded by one of the largest mammalian genes. The protein is formed by 27 domains, with 24 of them being single spectrin repeats interposed by two hinge regions. Single point and missing domain mutations within Dystrophin have been linked to Becker's Muscular Dystrophy (BMD), which is a degenerative muscle disease. This suggests that one role for Dystrophin is to reduce mechanical force between the sarcolemma membrane and the cytoskeleton. The mechanism for this function is unknown. The observation that single point mutations within Dystrophin are correlated with BMD suggests there is communication between each amino acid within each Dystrophin domain and that this communication extends to its neighboring domains. This led to the hypothesis that as force is transduced through the spectrin repeats, this results in the partial unfolding of the repeats which then dissipates force. If this partial unfolding of a given domain is accentuated when another repeat domain is in tandem, then this is consistent with negative coupling. Conversely, stabilization of one domain by another is positive coupling. Molecular dynamics (MD) forced unfolding experiments were consistent with negative coupling through non-additive force trajectories for tandem domains compared to individual domains. Therefore, we designed an experimental approach to test the computational results.Using monomeric spectrin 17, monomeric spectrin 18, and dimeric spectrin 17-18, we experimentally defined the thermodynamic and structural basis of energetic coupling between the spectrin domains through differential scanning calorimetry (DSC), fluorescence lifetime (FLT), and Overhauser dynamic nuclear polarization (ODNP). A combined approach utilizing these methods will provide insight into how coupling between spectrin repeats affects signal propagation and mechanical force dissipation. 1682-Pos Drug Resistance Induced by Local and Allosteric Conformational Changes in Oncogenic Tyrosine KinasesMitsugu Araki, Yasushi Okuno. Kyoto Univ, Kyoto, Japan. In non-small-cell lung cancer, the efficacies of tyrosine-kinase inhibitors (TKIs) are known to be decreased by mutations in target proteins such as ALK and RET, and in some cases there are no effective therapeutic strategies to overcome drug resistance. Here, we approach molecular mechanisms to acquire the resistance by combining molecular dynamics (MD) simulations on the nano-to micro-seconds timescales and our developed protein-ligand binding free energy computation method (Fujitani et al., Phys. Rev. E, (2009) 79, 021914 and Araki et al., J. Chem. Inf. Model., (2016) 56 (12), 2445-2456). Initial structures of mutants are modeled based on the crystal structures of the wild-type proteins, and their structural dynamics and binding affinities with TKIs are computed. In cases of drug resistance mutations in ALK, most of which occur near the drug binding site (< 10 angstroms), decreases in the efficacy tend to be successfully quantified by the binding free energy computatio...