Abdelaziz Alsamarah

Mechanism of gating by calcium in connexin hemichannels

Significance Connexin channels are ubiquitous, providing pathways for movement of molecules between cells (junctional channels) and for release of molecular effectors into the extracellular environment (plasma membrane hemichannels). To maintain an adequate permeability barrier, hemichannels are tightly regulated by normal extracellular Ca2+ to be closed under most conditions. Connexin mutations that disrupt hemichannel regulation by Ca2+ cause human pathologies due to aberrantly open hemichannels. Here we elucidate molecular mechanisms of gating by Ca2+ in hemichannels: Ca2+ binding causes a reorganization of specific interactions within the connexin protein that lead to a closed channel. Further, we show that the actual “gate” is deeper into the pore from where Ca2+ binds. The interactions involved are conserved across connexins, pointing to a general mechanism. Aberrant opening of nonjunctional connexin hemichannels at the plasma membrane is associated with many diseases, including ischemia and muscular dystrophy. Proper control of hemichannel opening is essential to maintain cell viability and is achieved by physiological levels of extracellular Ca2+, which drastically reduce hemichannel activity. Here we examined the role of conserved charged residues that form electrostatic networks near the extracellular entrance of the connexin pore, a region thought to be involved in gating rearrangements of hemichannels. Molecular dynamics simulations indicate discrete sites for Ca2+ interaction and consequent disruption of salt bridges in the open hemichannels. Experimentally, we found that disruption of these salt bridges by mutations facilitates hemichannel closing. Two negatively charged residues in these networks are putative Ca2+ binding sites, forming a Ca2+-gating ring near the extracellular entrance of the pore. Accessibility studies showed that this Ca2+-bound gating ring does not prevent access of ions or small molecules to positions deeper into the pore, indicating that the physical gate is below the Ca2+-gating ring. We conclude that intra- and intersubunit electrostatic networks at the extracellular entrance of the hemichannel pore play critical roles in hemichannel gating reactions and are tightly controlled by extracellular Ca2+. Our findings provide a general mechanism for Ca2+ gating among different connexin hemichannel isoforms.

Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity.

Abnormal alteration of bone morphogenetic protein (BMP) signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI) to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-β type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2) tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD) simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5) or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2), as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189) will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling.

Development of New Therapeutic Agents for Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP, MIM #135100) is a rare genetic disorder of heterotopic endochondral ossification, resulting in transformation of soft tissue into episodic bone formation. Currently, no effective treatment for FOP has been established. The causative heterozygous genetic mutations have been identified in either the intracellular glycine-serine-rich (GS) domain or kinase domain of ALK2 (Activin-like kinase-2, also known as Activin A receptor type I, ACVR1), a type I receptor of bone morphogenetic proteins (BMP). Cumulative studies support that these mutations abnormally activate BMP signaling in a ligandindependent manner by reducing the ALK2 interaction with the negative regulator FKBP12, whereas others argue a ligand-dependent BMP signaling activation in FOP. Nevertheless, in either the ligand-independent or ligand-dependent model, ALK2 receptor activation is essential for heterotopic ossification in FOP. Thus targeting ALK2 likely represents an effective treatment for FOP. In this article, we critically review the recent progress on therapeutic strategies, with a focus on development of small molecule ALK2 inhibitors to suppress BMP signaling for FOP treatment.

Polymodal allosteric regulation of Type 1 Serine/Threonine Kinase Receptors via a conserved electrostatic lock

Type 1 Serine/Threonine Kinase Receptors (STKR1) transduce a wide spectrum of biological signals mediated by TGF-β superfamily members. The STKR1 activity is tightly controlled by their regulatory glycine-serine rich (GS) domain adjacent to the kinase domain. Despite decades of studies, it remains unknown how physiological or pathological GS domain modifications are coupled to STKR1 kinase activity. Here, by performing molecular dynamics simulations and free energy calculation of Activin-Like Kinase 2 (ALK2), we found that GS domain phosphorylation, FKBP12 dissociation, and disease mutations all destabilize a D354-R375 salt-bridge, which normally acts as an electrostatic lock to prevent coordination of adenosine triphosphate (ATP) to the catalytic site. We developed a WAFEX-guided principal analysis and unraveled how phosphorylation destabilizes this highly conserved salt-bridge in temporal and physical space. Using current-flow betweenness scores, we identified an allosteric network of residue-residue contacts between the GS domain and the catalytic site that controls the formation and disruption of this salt bridge. Importantly, our novel network analysis approach revealed how certain disease-causing mutations bypass FKBP12-mediated kinase inhibition to produce leaky signaling. We further provide experimental evidence that this salt-bridge lock exists in other STKR1s, and acts as a general safety mechanism in STKR1 to prevent pathological leaky signaling. In summary, our study provides a compelling and unifying allosteric activation mechanism in STKR1 kinases that reconciles a large number of experimental studies and sheds light on a novel therapeutic avenue to target disease-related STKR1 mutants.

Can Relative Binding Free Energy Predict Selectivity of Reversible Covalent Inhibitors

Reversible covalent inhibitors have many clinical advantages over noncovalent or irreversible covalent drugs. However, apart from selecting a warhead, substantial efforts in design and synthesis are needed to optimize noncovalent interactions to improve target-selective binding. Computational prediction of binding affinity for reversible covalent inhibitors presents a unique challenge since the binding process consists of multiple steps, which are not necessarily independent of each other. In this study, we lay out the relation between relative binding free energy and the overall reversible covalent binding affinity using a two-state binding model. To prove the concept, we employed free energy perturbation (FEP) coupled with λ-exchange molecular dynamics method to calculate the binding free energy of a series of α-ketoamide analogues relative to a common warhead scaffold, in both noncovalent and covalent binding states, and for two highly homologous proteases, calpain-1 and calpain-2. We conclude that covalent binding state alone, in general, can be used to predict reversible covalent binding selectivity. However, exceptions may exist. Therefore, we also discuss the conditions under which the noncovalent binding step is no longer negligible and propose to combine the relative FEP calculations with a single QM/MM calculation of warhead to predict the binding affinity and binding kinetics. Our FEP calculations also revealed that covalent and noncovalent binding states of an inhibitor do not necessarily exhibit the same selectivity. Thus, investigating both binding states, as well as the kinetics will provide extremely useful information for optimizing reversible covalent inhibitors.

Interaction of Resveratrol with Lipid Membranes

Background: Resveratrol is a phytoalexin synthesized by plants. It has antioxidant properties and is a popular nutritional supplement. Beneficial properties of resveratrol, such as, anticancer and anti-inflammatory properties have been reported. Resveratrol is a constituent of red wine and found in the skin of red grapes. In order to understand the interaction between resveratrol and biological membranes, we evaluated the effect of resveratrol on model lipid membranes using differential scanning calorimetry (DSC) and computational studies. Methods: Phospholipids such as, Dilauroylphosphatidylcholine (DLPC), Dimyristoyphosphatidylcholine (DMPC), Dipalmitoylphosphatidyl choline (DPPC), Distearoylphosphatidylcholine (DSPC), and 1-palmitoyl-2-oleyl phosphatidylcholine (POPC) were purchased from Avanti Polar Lipids (Alabaster, Alabama). Each phospholipid was mixed with resveratrol at different molar ratios, phospholipid: resveratrol, 10:1, 10:3 and 10:5. Computational simulations were also performed to evaluate the interactions of DSPC and resveratrol.Results: Resveratrol abolishes only the transition of the DLPC, which is the shortest phospholipid of those tested. It reduces the transition temperature for all the other phospholipids even at the lowest ratio tested, phospholipid: resveratrol, 10:1. Resveratrol reduces the transition temperature of DSPC from 55 °C to 51 °C. Furthermore, using DSC, we also observed another transition, a sharp exothermic peak above 275 °C in the interaction of resveratrol with DSPC. We performed computational simulations of the DSPC membrane at different temperatures with and without resveratrol. The simulation indicates that resveratrol affects the transition temperature of the DSPC, which is in agreement with our DSC data. In conclusion, our data indicate that resveratrol abolishes the transition of DLPC and acts as a plasticizer for phospholipids with longer fatty acyl chains.