The best-known mode of action of calmodulin (CaM) is binding of Ca 2 + to its N- and C-domains, followed by binding to target proteins. An underappreciated facet of this process is that CaM is typically bound to proteins at basal levels of free Ca 2 + , including the small, intrinsically disordered, neuronal IQ-motif proteins called PEP -19 and neurogranin (Ng). PEP -19 and Ng would not be effective competitive inhibitors of high-affinity Ca 2 + -dependent CaM targets at equilibrium because they bind to CaM with relatively low affinity, but they could influence the time course of CaM signaling by affecting the rate of association of CaM with high-affinity Ca 2 + -dependent targets. This mode of regulation may be domain specific because PEP -19 binds to the C-domain of CaM, whereas Ng binds to both N- and C-domains. In this report, we used a model CaM binding peptide (CKIIp) to characterize the preferred pathway of complex formation with Ca 2 + -CaM at low levels of free Ca 2 + (0.25-1.5 mM), and how PEP19 and Ng affect this process. We show that the dominant encounter complex involves association of CKIIp with the N-domain of CaM, even though the C-domain has a greater affinity for Ca 2 + . We also show that Ng greatly decreases the rate of association of Ca 2 + -CaM with CKIIp due to the relatively slow dissociation of Ng from CaM, and to interactions between the Gly-rich C -terminal region of Ng with the N-domain of CaM, which inhibits formation of the preferred encounter complex with CKIIp. These results provide the general mechanistic paradigms that binding CaM to targets can be driven by its N-domain, and that low-affinity regulators of CaM signaling have the potential to influence the rate of activation of high-affinity CaM targets and potentially affect the distribution of limited CaM among multiple targets during Ca 2 + oscillations. SIGNIFICANCE Calmodulin is a small, essential regulator of multiple cellular processes including growth and differentiation. Its best-known mode of action is to first bind calcium and then bind and regulate the activity of target proteins. Each domain of calmodulin has distinct calcium binding properties and can interact with targets in distinct ways. Here, we show that the N-domain of calmodulin can drive its association with targets, and that a small, intrinsically disordered regulator of calmodulin signaling called neurogranin can greatly decrease the rate of association of calmodulin with high-affinity Ca 2 + -dependent targets. These results demonstrate the potential of neurogranin, and other proteins, to modulate the time course of activation of targets by a limited intracellular supply of calmodulin.

We used NMR to show that the antipsychotic phenothiazine drugs promazine and promethazine bind to GDP-KRAS. Promazine also binds to oncogenic GDP-KRAS(G12D), and to wild type GppNHp-KRAS. A panel of additional phenothiazines bind to GDP-KRAS but with lower affinity than promazine or promethazine. Binding is most dependent on substitutions at C-2 of the tricyclic phenothiazine ring. Promazine was used to generate an NMR-driven HADDOCK model of the drug/GDP-KRAS complex. The structural model shows the tricyclic phenothiazine ring of promazine associates with the hydrophobic pocket p1 that is bordered by the central β sheet and Switch II in KRAS. Binding appears to stabilize helix 2 in a conformation that is similar to that seen in KRAS bound to other small molecules. Association of phenothiazines with KRAS may affect normal KRAS signaling that could contribute to multiple biological activities of these antipsychotic drugs. Moreover, the phenothiazine ring represents a new core scaffold on which to design modulators of KRAS activity.

RAS mutations account for >15% of all human tumors, and of these ~85% are due to mutations in a particular RAS gene: KRAS. Recent studies revealed that KRAS harbors four druggable allosteric sites. Here, we have (a) used molecular simulations to generate ensembles of wild type and four major oncogenic KRAS mutants (G12V, G12D, G13D, and Q61H); (b) characterized the druggability of each allosteric pocket in each protein; (c) conducted extensive ensemble‐based virtual screening using pocket‐tailored ligand libraries; (d) prioritized hits through hierarchical postdocking analysis; and (e) validated predicted hits with NMR. Of the 785 diverse potential hits identified by our in silico analysis, we tested 90 for their ability to bind KRAS using NMR and found that nine cause backbone amide chemical shift perturbations of residues near the functionally responsive switch loops, suggesting potential binding. We conducted detailed biophysical analyses on a novel indole‐based compound to demonstrate the potential of our workflow to yield lead compounds. We believe the detailed information documented in this work regarding the druggability profile of each allosteric site and the chemical fingerprints of compounds that target them will serve as vital resources for future structure‐based drug design efforts against KRAS, a high‐value target for cancer therapy.

Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction, first cleaving the LPXTG motif of one pilin protomer to form an acylenzyme intermediate and then joining the terminal Thr to the nucleophilic Lys residue residing within the pilin motif of another pilin protomer. To date, the determinants of class C enzymes that uniquely enable them to construct pili remain unknown. Here, informed by high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and utilizing a structural variant of the enzyme (SrtA(2M)), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Mass spectrometry, electron microscopy, and biochemical experiments authenticated that SrtA(2M) synthesizes pilus fibers with correct Lys-Thr isopeptide bonds linking individual pilins via a thioacyl intermediate. Structural modeling of the SpaA-SrtA-SpaA polymerization intermediate depicts SrtA(2M) sandwiched between the N- and C-terminal domains of SpaA harboring the reactive pilin and LPXTG motifs, respectively. Remarkably, the model uncovered a conserved TP(Y/L)XIN(S/T)H signature sequence following the catalytic Cys, in which the alanine substitutions abrogated cross-linking activity but not cleavage of LPXTG. These insights and our evidence that SrtA(2M) can terminate pilus polymerization by joining the terminal pilin SpaB to SpaA and catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile platform for protein engineering and bio-conjugation that has major implications for biotechnology.

K-Ras is a small GTPase that plays a critical role in the regulation of a variety of signaling pathways involved in cell growth and proliferation. Somatic mutations on K-Ras are associated with many different cancers, accounting for about 85% of all Ras-associated cancers or 15-20% of all human cancers. K-Ras is a very dynamic allosteric enzyme and our previous studies revealed that K-Ras harbors four allosteric ligand-binding pockets. This suggested that targeting K-Ras directly is a viable strategy to abrogate its abnormal functions. In the current study, we conducted extensive multi conformer hierarchical virtual screening to identify potential hits targeting each of these four pockets. In the first step we used clustering, structure-based pocket analysis and knowledge-based filters on conformers obtained from all-atom MD simulations of oncogenic mutant K-Ras to select representative structures for docking. The representative structures were further analyzed in terms of their physico-chemical properties to gain an insight into the unique features of each pocket. Based on this the large purchasable chemical space of the ZINC database was tailored to generate complementary pocket-specific chemical libraries. We performed ensemble docking of these tailored ligand libraries against each pocket with the standard precision module of the Glide docking software, and used hierarchical post-docking analysis (PDA) to identify plausible K-Ras binders. In PDA the docking outputs of ligands were grouped based on their common residues interaction pattern (A) and chemical scaffold diversity (B) at the each binding site. A primary list of highest scoring hits per pocket was made from the ligands in the B subgroup that belonged to subgroup A, followed by visual inspection of the binding pose. This yielded a list of 761 potential hits. 217 of these were procured for experimental testing and about 100 were tested for their ability to bind to GDP-bound K-Ras using N15-labeled heteronuclear single quantum coherence (HSQC) NMR. Of the 100 tested, 11 showed significant chemical shift perturbations at several residues, suggesting potential binding. We will discuss these results and their implications for future efforts in K-Ras drug discovery.

Increasing evidence has demonstrated that small nucleolar RNAs (snoRNAs) play important roles in tumorigenesis. We systematically investigated the expression landscape and clinical relevance of snoRNAs in > 10,000 samples across 31 cancer types from The Cancer Genome Atlas. We observed overall elevated expression of snoRNAs and their ribonucleoproteins in multiple cancer types. We showed complex regulation of snoRNA expression by their host genes, copy number variation, and DNA methylation. Unsupervised clustering revealed that the snoRNA expression subtype is highly concordant with other molecular/clinical subtypes. We further identified 46 clinically relevant snoRNAs and experimentally demonstrated functional roles of SNORD46 in promoting cell proliferation, migration, and invasion. We developed a user-friendly data portal, SNORic, to benefit the research community. Our study highlights the significant roles of snoRNAs in the development and implementation of biomarkers or therapeutic targets for cancer and provides a valuable resource for cancer research.

PEP-19 is a small protein that increases the rates of Ca2+ binding to the C-domain of calmodulin (CaM) by an unknown mechanism. Although an IQ motif promotes binding to CaM, an acidic sequence in PEP-19 is required to modulate Ca2+ binding and to sensitize HeLa cells to ATP-induced Ca2+ release. Here, we report the NMR solution structure of a complex between PEP-19 and the C-domain of apo CaM. The acidic sequence of PEP-19 associates between helices E and F of CaM via hydrophobic interactions. This allows the acidic side chains in PEP-19 to extend toward the solvent and form a negatively charged surface that resembles a catcher's mitt near Ca2+ binding loop III of CaM. The topology and gradients of negative electrostatic surface potential support a mechanism by which PEP-19 increases the rate of Ca2+ binding to the C-domain of CaM by 'catching' and electrostatically steering Ca2+ to site III.

Protein signaling occurs in crowded intracellular environments, and while high concentrations of macromolecules are postulated to modulate protein-protein interactions, analysis of their impact at each step of the reaction pathway has not been systematically addressed. Potential cosolute-induced alterations in target association are particularly important for a signaling molecule like calmodulin (CaM), where competition among >300 targets governs which pathways are selectively activated. To explore how high concentrations of cosolutes influence CaM-target affinity and kinetics, we methodically investigated each step of the CaM-target binding mechanism under crowded or osmolyte-rich environments mimicked by ficoll-70, dextran-10, and sucrose. All cosolutes stabilized compact conformers of CaM and modulated association kinetics by affecting diffusion and rates of conformational change; however, the results showed that differently sized molecules had variable effects to enhance or impede unique steps of the association pathway. On- and off-rates were modulated by all cosolutes in a compensatory fashion, producing little change in steady-state affinity. From this work insights were gained on how high concentrations of inert crowding agents and osmolytes fit into a kinetic framework to describe protein-protein interactions relevant for cellular signaling.

1071-Pos Board B22 Targeting Melanoma with Small Molecules: Inhibitors of the CalciumBinding Protein S100B Michael C. Cavalier, David J. Weber. Center for Biomolecular Therapeutics, University of Maryland School of Medicine, Baltimore, MD, USA. Long used as a prognostic indicator, the calcium binding protein, S100B, has been directly linked to Malignant Melanoma (MM). S100B binds the typically wild-type p53 tumor suppressor in MM and promotes its degradation. In order to restore p53 levels within MM along with its tumor suppressor and apoptotic activities, we have implemented a program for the discovery and optimization of S100B inhibitors (often referred to as SBiXs). In the process, we have uncovered and probed the three persistent binding sites within S100B. Liganding within these sites was characterized using structural biology techniques (NMR and X-ray crystallography) and inhibitor efficacy was evaluated using Fluorescent Polarization Competition Assays and cellular assays. Efforts to discover/synthesize and/or to improve existing inhibitors of S100B to restore p53 activity in human malignant melanoma are ongoing and SBiXs occupying the persistent binding Sites 1, 2, and 3 simultaneously are desired. Such compounds can then be examined for in vivo efficacy. The compounds presented offer potential bridging scaffolds between Sites 1, 2, and 3; and will act as the basis for the design of improved SBiXs. SBiXs may also have therapeutic value for treating other cancers with elevated S100B and wt p53 such as astrocytoma, renal tumors, and some forms of leukemia.

Background: PEP-19 has no known intrinsic activity other than binding predominately to the C-domain of calmodulin (C-CaM), yet it is implicated in numerous cellular processes. We showed that an acidic sequence in PEP-19 is required to greatly increase the rates of Ca2+ binding to C-CaM. Importantly, the acidic sequence is also required for PEP-19 to sensitize HeLa cells to ATP-induced Ca2+ release. Goal: The goal of the current study was to determine the high-resolution NMR solution structure of the PEP-19/apo C-CaM complex. Results: Apo C-CaM adopts a semi-open conformation when bound to PEP-19, with the helices E and F of Ca2+ binding site III showing the greatest change in angle relative to free apo C-CaM. The conformation of Ca2+ binding loop III is similar to that in free apo C-CaM, but loop IV adopts a different conformation with increased conformational exchange when bound to PEP-19. Residues 1-29 in PEP-19 remain disordered when bound to C-CaM, and are thus accessible for potential interactions with other proteins. The IQ motif in PEP-19 adopts a well-defined alpha helix that binds to a hydrophobic groove in apo C-CaM. The C-terminal part of the acidic sequence is alpha helical, but the N-terminal portion (aa 28-36) forms loop and coil structures that are stabilized by interactions between Ile32 and Met34 in PEP-19 and hydrophobic residues in Ca2+ binding site III of C-CaM. This allows acid side chains in PEP-19 to extend toward the solvent to greatly increase negative charge density near site III of C-CaM. Conclusions: The structure suggests that the acidic sequence in PEP-19 modulates Ca2+ binding to site III of C-CaM by direct interactions and/or electrostatic steering of Ca2+, but that allosteric effects increase conformational exchange to modulate Ca2+ binding to site IV.

The cellular interior is crowded with high concentrations of macromolecules and small organic solutes and strict maintenance of this environment, where ∼30% of the total volume is occupied, is essential to cellular function. The volume occupied by these molecules confines the 3D space in which any particular protein can reside and is thought to modulate protein-protein interactions by affecting diffusional encounter and reaction rates. In this work, we investigate the mechanistic consequences of crowded environments on the structure-function relationship of the ubiquitous protein calmodulin (CaM). CaM is highly flexible and dynamic, making it particularly susceptible to crowded environments, and its conformational plasticity is essential for accommodating binding to its 300 identified targets. We determined that crowded environments, created with purified polymer systems and sucrose, stabilize compact conformers of CaM, reduce translational and rotational diffusion, modulate association and dissociation kinetics with a calmodulin-binding target, and control rates of conformational transition. Most notably, these effects are determined by the size and concentration of the crowding reagent indicating that the composition of the environment differentially tunes CaM structure, dynamics, and target association kinetics. Our results are shown in a general kinetic framework where there are consequences for target selectivity and cellular signaling.

response to stress. Yet, despite its obvious importance, little is known regarding even the identity (or the function) of the intracellular molecular pathways underpinning the trafficking and targeting of integral membrane proteins in the context of the native heart. The goal of this work is identify new molecular players that regulate protein targeting and trafficking in the heart. Eps 15 homology domain-containing (EHD) gene products (EHD1-4) are intracellular proteins that are key regulators of endosomal trafficking, lipid homeostasis, membrane protein recycling and trafficking. Previously uncharacterized in the heart, we recently presented evidence which demonstrated that this protein family likely plays indispensable roles in protein trafficking in cardiac muscle. Notably, an essential role for one of these proteins, EHD3, in the membrane trafficking of the Na/Ca exchanger (NCX) in heart was uncovered. The goal of this research program is for the first time to directly test the role of these proteins in cardiac structural and electrical activity using cutting-edge in vivo models of EHD deficiency. We show that EHD3 deficiency in the heart leads to: 1) abnormal cardiac structure at baseline; 2) irregular action potential morphology, heart rhythm, and conduction; 3) depressed b-adrenergic responsiveness; 4) dysfunctional NCX and LTCC trafficking and function; and 5) dysregulated ankyrin-B expression and trafficking. These data strongly support a role for EHD3 in membrane protein trafficking and regulation within the context of the native heart.

We aimed to elucidate the role of protein dynamics and stability in target recognition, a process critical in understanding how proteins "choose" the appropriate target. We chose to examine the ubiquitous protein calmodulin (CaM), an essential secondary messenger of calcium signaling with over 300 identified protein targets. CaM is highly flexible and dynamic and its conformational plasticity is essential for accommodating diverse targets. CaM exists as an ensemble of conformers fluctuating around a large flat energy minimum, where conformational sampling permits a continuum population of structures with similar energies. To investigate the role of conformational sampling we measured CaM properties under conditions of excluded volume, where non-reactive polymers were incorporated into experimental conditions. The polymers effectively limit the 3D space a protein can occupy and thus influence its conformational sampling by redistributing the probabilities of each conformational state and modulating the energy barriers between them. We observed that volume exclusion stabilized compact conformations of CaM which had minimal impact on steady state affinity for Ca2+ or for protein targets. We observed reduced association rates with targets and importantly, volume exclusion significantly decreased the rate of conformational transition from the initial encounter complex to the natively bound complex. This transition is dependent on polymer size, with smaller polymers correlated to larger decreases, as well as temperature, where lower temperatures which impede conformational dynamics decreased rates the most significantly. We conclude that stabilization of compact conformers and dampened protein dynamics induce conformational frustration during transition to the native complex, where sampling of productive transition state intermediates is encumbered by high energy barriers. Our data is in support of an induced fit binding mechanism where CaM and its target must undergo mutually adjusted step-wise conformational searches to find the natively bound complex.

Neurogranin (Ng) is a member of the IQ motif class of calmodulin (CaM)-binding proteins, and interactions with CaM are its only known biological function. In this report we demonstrate that the binding affinity of Ng for CaM is weakened by Ca2+ but to a lesser extent (2-3-fold) than that previously suggested from qualitative observations. We also show that Ng induced a > 10-fold decrease in the affinity of Ca2+ binding to the C-terminal domain of CaM with an associated increase in the Ca2+ dissociation rate. We also discovered a modest, but potentially important, increase in the cooperativity in Ca2+ binding to the C-lobe of CaM in the presence of Ng, thus sharpening the threshold for the C-domain to become Ca2+-saturated. Domain mapping using synthetic peptides indicated that the IQ motif of Ng is a poor mimetic of the intact protein and that the acidic sequence just N-terminal to the IQ motif plays an important role in reproducing Ng-mediated decreases in the Ca2+ binding affinity of CaM. Using NMR, full-length Ng was shown to make contacts largely with residues in the C-domain of CaM, although contacts were also detected in residues in the N-terminal domain. Together, our results can be consolidated into a model where Ng contacts residues in the N- and C-lobes of both apo- and Ca2+-bound CaM and that although Ca2+ binding weakens Ng interactions with CaM, the most dramatic biochemical effect is the impact of Ng on Ca2+ binding to the C-terminal lobe of CaM.

Background: Neurogranin (Ng) and PEP-19 are small proteins with no known intrinsic activity other than binding to calmodulin (CaM) via their IQ motifs, yet they have been implicated in numerous normal and pathological processes. We showed that PEP-19 is intrinsically disordered, and that an acidic sequence adjacent to its IQ motif is required for PEP-19 to modulate Ca2+ binding to CaM, and to sensitize HeLa cells to ATP-induced Ca2+ release. Goals: Ng has an acidic sequence, but with significantly different composition than PEP-19. Thus, the goals of the current study were to determine: 1) If the acidic sequence in Ng is required to modulate Ca2+ binding to CaM; and 2) Use NMR to compare the effects of Ng derivatives on the conformation of CaM. Results: Ng greatly increases the Ca2+ koff at the C-domain of CaM, but has little effect on the kon, thereby decreasing Ca2+ binding affinity. The peptide Ng(29-49), which includes only the consensus IQ motif does not increase the Ca2+ koff, but Ng(13-49), which includes the acidic region of Ng mimics the effect of intact Ng on Ca2+ binding to CaM. The 1H 15N HSQC spectra of Ng show it to be an intrinsically disordered protein. Effects of Ng protein and peptides on the NMR spectra of CaM are consistent with their relative effects on Ca2+ binding. Also, effects of Ng (29-49) on amide chemical shift perturbations and backbone dynamic properties of CaM are different from Ng or Ng(13-49), especially in the apo state. Conclusions: Modulating Ca2+ binding to CaM relies on the acidic region of both PEP-19 and Ng. Tuning Ca2+ mobilization pathways by PEP-19 and Ng expands the biological significance of these intrinsically disordered regulators of CaM signaling.

Calmodulin, an intracellular calcium-binding protein, is thought to regulate ectodomain shedding of many membrane proteins, but the underlying molecular mechanism has remained unclear. Basing on a solution structure of calcium-loaded calmodulin in complex with a L-selectin fragment that contains a portion of its transmembrane domain, Gifford et al. (University of Calgary) recently suggested that calmodulin regulates L-selectin shedding by binding directly to a portion of the L-selectin transmembrane domain in a compact conformation. Using fluorescently labeled calmodulin, we show however that calmodulin adopts a distinctly different and much more extended conformation when it binds to the CLS peptide (i.e. the entire transmembrane and cytoplasmic domains of L-selectin) reconstituted in the phosphatidylcholine liposome with micromolar dissociation constant and in a calcium-independent manner. Calmodulin adopts a similarly extended conformation in a ternary complex with the N-terminal FERM domain of moesin and CLS reconstituted in the phospholipid liposome that mimics the native membrane environment. These results indicate that calmodulin does not bind directly to the transmembrane domain of L-selectin. Understanding the association of calmodulin with L-selectin helps to shed light on the mechanisms underlying regulation of ectodomain shedding.

PEP-19 is a small, intrinsically disordered protein that binds to the C-domain of calmodulin (CaM) via an IQ motif and tunes its Ca2+ binding properties via an acidic sequence. We show here that the acidic sequence of PEP-19 has intrinsic Ca2+ binding activity, which may modulate Ca2+ binding to CaM by stabilizing an initial Ca2+-CaM complex or by electrostatically steering Ca2+ to and from CaM. Because PEP-19 is expressed in cells that exhibit highly active Ca2+ dynamics, we tested the hypothesis that it influences ligand-dependent Ca2+ release. We show that PEP-19 increases the sensitivity of HeLa cells to ATP-induced Ca2+ release to greatly increase the percentage of cells responding to sub-saturating doses of ATP and increases the frequency of Ca2+ oscillations. Mutations in the acidic sequence of PEP-19 that inhibit or prevent it from modulating Ca2+ binding to CaM greatly inhibit its effect on ATP-induced Ca2+ release. Thus, this cellular effect of PEP-19 does not depend simply on binding to CaM via the IQ motif but requires its acidic metal binding domain. Tuning the activities of Ca2+ mobilization pathways places PEP-19 at the top of CaM signaling cascades, with great potential to exert broad effects on downstream CaM targets, thus expanding the biological significance of this small regulator of CaM signaling.