Signal Transduction and Pathogenic Modifications at the Melanocortin-4 Receptor: A Structural Perspective

The melanocortin-4 receptor (MC4R) can be endogenously activated by binding of melanocyte-stimulating hormones (MSH), which mediates anorexigenic effects. In contrast, the agouti-related peptide (AgRP) acts as an endogenous inverse agonist and suppresses ligand-independent basal signaling activity (orexigenic effects). Binding of ligands to MC4R leads to the activation of different G-protein subtypes or arrestin and concomitant signaling pathways. This receptor is a key protein in the hypothalamic regulation of food intake and energy expenditure and naturally-occurring inactivating MC4R variants are the most frequent cause of monogenic obesity. In general, obesity is a growing problem on a global scale and is of social, medical, and economic relevance. A significant goal is to develop optimized pharmacological tools targeting MC4R without adverse effects. To date, this has not been achieved because of inter alia non-selective ligands across the five functionally different MCR subtypes (MC1-5R). This motivates further investigation of (i) the three-dimensional MC4R structure, (ii) binding mechanisms of various ligands, and (iii) the molecular transfer process of signal transduction, with the aim of understanding how structural features are linked with functional-physiological aspects. Unfortunately, experimentally elucidated structural information is not yet available for theMC receptors, a group of class A G-protein coupled receptors (GPCRs). We, therefore, generated MC4R homology models and complexes with interacting partners to describe approximate structural properties associated with signaling mechanisms. In addition, molecular insights from pathogenic mutations were incorporated to discriminate more precisely their individual malfunction of the signal transfer mechanism

Conformational perturbation, allosteric modulation of cellular signaling pathways, and disease in P23H rhodopsin

In this investigation we use THz spectroscopy and MD simulation to study the functional dynamics and conformational stability of P23H rhodopsin. The P23H mutation of rod opsin is the most common cause of human binding autosomal dominant retinitis pigmentosa (ADRP), but the precise mechanism by which this mutation leads to photoreceptor cell degeneration has not yet been elucidated. Our measurements confirm conformational instability in the global modes of the receptor and an activestate that uncouples the torsional dynamics of the retinal with protein functional modes, indicating inefficient signaling in P23H and a drastically altered mechanism of activation when contrasted with the wild-type receptor. Further, our MD simulations indicate that P23H rhodopsin is not functional as a monomer but rather, due to the instability of the mutant receptor, preferentially adopts a specific homodimerization motif. The preferred homodimer configuration induces structural changes in the receptor tertiary structure that reduces the affinity of the receptor for the retinal and significantly modifies the interactions of the Meta-II signaling state. We conjecture that the formation of the specific dimerization motif of P23H rhodopsin represents a cellular-wide signaling perturbation that is directly tied with the mechanism of P23H disease pathogenesis. Our results also support a direct role for rhodopsin P23H dimerization in photoreceptor rod death

Computational Modeling of Allosteric Communication Reveals Organizing Principles of Mutation-Induced Signaling in ABL and EGFR Kinases

The emerging structural information about allosteric kinase complexes and the growing number of allosteric inhibitors call for a systematic strategy to delineate and classify mechanisms of allosteric regulation and long-range communication that control kinase activity. In this work, we have investigated mechanistic aspects of long-range communications in ABL and EGFR kinases based on the results of multiscale simulations of regulatory complexes and computational modeling of signal propagation in proteins. These approaches have been systematically employed to elucidate organizing molecular principles of allosteric signaling in the ABL and EGFR multi-domain regulatory complexes and analyze allosteric signatures of the gate-keeper cancer mutations. We have presented evidence that mechanisms of allosteric activation may have universally evolved in the ABL and EGFR regulatory complexes as a product of a functional cross-talk between the organizing αF-helix and conformationally adaptive αI-helix and αC-helix. These structural elements form a dynamic network of efficiently communicated clusters that may control the long-range interdomain coupling and allosteric activation. The results of this study have unveiled a unifying effect of the gate-keeper cancer mutations as catalysts of kinase activation, leading to the enhanced long-range communication among allosterically coupled segments and stabilization of the active kinase form. The results of this study can reconcile recent experimental studies of allosteric inhibition and long-range cooperativity between binding sites in protein kinases. The presented study offers a novel molecular insight into mechanistic aspects of allosteric kinase signaling and provides a quantitative picture of activation mechanisms in protein kinases at the atomic level.This work was partly supported by funding from The University of Kansas

Allo-network drugs: Extension of the allosteric drug concept to protein-protein interaction and signaling networks

Allosteric drugs are usually more specific and have fewer side effects than orthosteric drugs targeting the same protein. Here, we overview the current knowledge on allosteric signal transmission from the network point of view, and show that most intra-protein conformational changes may be dynamically transmitted across protein-protein interaction and signaling networks of the cell. Allo-network drugs influence the pharmacological target protein indirectly using specific inter-protein network pathways. We show that allo-network drugs may have a higher efficiency to change the networks of human cells than those of other organisms, and can be designed to have specific effects on cells in a diseased state. Finally, we summarize possible methods to identify allo-network drug targets and sites, which may develop to a promising new area of systems-based drug design

Phosphorylation of the Arp2 subunit relieves auto-inhibitory interactions for Arp2/3 complex activation.

Actin filament assembly by the actin-related protein (Arp) 2/3 complex is necessary to build many cellular structures, including lamellipodia at the leading edge of motile cells and phagocytic cups, and to move endosomes and intracellular pathogens. The crucial role of the Arp2/3 complex in cellular processes requires precise spatiotemporal regulation of its activity. While binding of nucleation-promoting factors (NPFs) has long been considered essential to Arp2/3 complex activity, we recently showed that phosphorylation of the Arp2 subunit is also necessary for Arp2/3 complex activation. Using molecular dynamics simulations and biochemical assays with recombinant Arp2/3 complex, we now show how phosphorylation of Arp2 induces conformational changes permitting activation. The simulations suggest that phosphorylation causes reorientation of Arp2 relative to Arp3 by destabilizing a network of salt-bridge interactions at the interface of the Arp2, Arp3, and ARPC4 subunits. Simulations also suggest a gain-of-function ARPC4 mutant that we show experimentally to have substantial activity in the absence of NPFs. We propose a model in which a network of auto-inhibitory salt-bridge interactions holds the Arp2 subunit in an inactive orientation. These auto-inhibitory interactions are destabilized upon phosphorylation of Arp2, allowing Arp2 to reorient to an activation-competent state

Structural Dynamics and Allosteric Signaling in Ionotropic Glutamate Receptors

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitatory neurotransmission events in the central nervous system. All distinct classes of iGluRs (AMPA, NMDA, Kainate) are composed of an N-terminal domain (NTD) and a ligand-binding domain (LBD) in their extracellular domain, a transmembrane domain (TMD) and an intracellular carboxy-terminal domain (CTD). Ligand binding to the LBD facilitates ion channel activation. The NTDs modulate channel gating allosterically in NMDA receptors (NMDARs). A similar function of the NTD in AMPA receptors (AMPARs) is still a matter of debate. Taking advantage of recently resolved structures of the NTD and the intact AMPAR, the main focus of this dissertation is a comprehensive examination of iGluR NTD structural dynamics, ligand binding and allosteric potential of AMPARs. We use a multiscale, multi-dimensional approach using coarse-grained network models and all-atom simulations for structural analyses and information theoretic approaches for examination of evolutionary correlations. Our major contribution has been the characterization of the global motions favored by iGluR NTD architecture. These intrinsic motions favor ligand binding in NMDAR NTDs and are also shared by other iGluR NTDs. We also identified structural determinants of flexibility in AMPARs and confirmed their role through in silico mutants. The overall similarity in collective dynamics among iGluRs hints at a putative allosteric capacity of non-NMDARs and has propelled the elucidation of interdomain and intersubunit coupling in the intact AMPAR. To this end, we identified “effector” and “sensor” regions in AMPARs using a perturbation-response technique. We identified potentially functional residues that enable information propagation between effector regions and proposed an efficient mechanism of allosteric communication based on a combination of tools including network models, graph theoretical methods and sequence analyses. Finally, we assessed the “druggability” of iGluR NTDs using molecular dynamics simulations in the presence of probe molecules containing fragments shared by drug-like molecules. Based on our study, we offer key insights into the ligand-binding landscape of iGluR NTD monomers and dimers, and we also identify a novel ligand-binding site in AMPAR dimers. These findings open an avenue of searching for molecules able to bind to iGluR NTDs and allosterically modulate receptor activity

MOLECULAR PHYSIOLOGY OF BLOOD-BRAIN BARRIER TIGHT JUNCTIONS

The molecular interface of the blood-brain barrier (BBB) is a highly selective physiological barrier. The BBB shields the central nervous system (CNS) for harmful agents while also preventing lifesaving drugs from entering the CNS. With the prevalence of neurogenerative disease on the rise, there is a growing interest to design therapeutic interventions that can surpass the BBB. Such efforts necessitate a thorough understanding of the BBB, requiring one to decipher: why the BBB is so selective? what governing molecular rules govern selectivity across the BBB? and how does it impact physiology. As a contribution towards this understanding the following dissertation discusses nuances of the BBB see from the perspective of its tight junctions (TJ). Tight junctions are a protein-protein adhesion structures that seal the paracellular space for small solutes. Tight junctions are a common feature in many epithelial and endothelial tissues and a crucial component of the BBB. The BBB tight junctions are shown to be regulate a size and charge selective barrier that permeates only molecules of 800 Da in size. In the following chapters a computational microscopy approach was utilized to probe different structural and biochemical features of the tight junction. Chapter 2 discusses the molecular assembly of tight junction proteins investigates for the first time under molecular dynamics simulations. The key findings included the discovery of dimeric interfaces that are seen to form tight structural contacts between conserved residues. An experimental investigation with formaldehyde as a cross-linker in HeLa cells validated the existence of such contacts. Chapter 3 investigated the tight junction assembly in the paracellular space of adjacent cells by mimicking this interface with two membranes. These simulations revealed the structural aspects of the pores that are feasible under claudin-5 tight junction assembly. We performed a mutation experiment that distinguished the dimeric interfaces between claudin-3 and claudin-5, further a biophysical investigation showed how the flexibility of the transmembrane domains affect the dimerization of claudins. Chapter 4 extends upon the discoveries from chapters 2 and 3 to other claudins that are relevant for the tight junction biology. There is an inherent need to compare different members of the claudin family of proteins to enhance the overall understanding about tight junction biology and consequently the BBB tight junctions. Major findings include the discovery of a putative trimeric receptor assembly for Clostridium perfringens enterotoxin. The pore assemblies of claudin-2 and the dynamics of ions across the pores. Chapter 5 investigates the ion selectivity of claudin-5 and claudin-2 in a greater detail. The key findings include that the barrier to charge selectivity in the claudin pores are due to charge repulsion from the pore lining residues. The electrostatic interaction dominates the pore selectivity while the steric interaction plays a role for divalent cations. These biophysical evidence reveal how the claudin-5 tight junction pores that line the BBB screen charged ions and water. These computational findings push the boundaries of current knowledge on the BBB and sets the stage for applications targeted towards drug delivery strategies. The computational methods and tools discussed herein sets precedent for its transferability to the investigation of other tight junction proteins and in wider scope other membrane proteins

Potential application of network descriptions for understanding conformational changes and protonation states of ABC transporters.

The ABC (ATP Binding Cassette) transporter protein superfamily comprises a large number of ubiquitous and functionally versatile proteins conserved from archaea to humans. ABC transporters have a key role in many human diseases and also in the development of multidrug resistance in cancer and in parasites. Although a dramatic progress has been achieved in ABC protein studies in the last decades, we are still far from a detailed understanding of their molecular functions. Several aspects of pharmacological ABC transporter targeting also remain unclear. Here we summarize the conformational and protonation changes of ABC transporters and the potential use of this information in pharmacological design. Network related methods, which recently became useful tools to describe protein structure and dynamics, have not been applied to study allosteric coupling in ABC proteins as yet. A detailed description of the strengths and limitations of these methods is given, and their potential use in describing ABC transporter dynamics is outlined. Finally, we highlight possible future aspects of pharmacological utilization of network methods and outline the future trends of this exciting field