Molecular Mechanism for Inhibition of G Protein-Coupled Receptor Kinase 2 by a Selective RNA Aptamer

SummaryCardiovascular homeostasis is maintained in part by the rapid desensitization of activated heptahelical receptors that have been phosphorylated by G protein-coupled receptor kinase 2 (GRK2). However, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. We have determined crystallographic structures of GRK2 bound to an RNA aptamer that potently and selectively inhibits kinase activity. Key to the mechanism of inhibition is the positioning of an adenine nucleotide into the ATP-binding pocket and interactions with the basic αF-αG loop region of the GRK2 kinase domain. Constraints imposed on the RNA by the terminal stem of the aptamer also play a role. These results highlight how a high-affinity aptamer can be used to selectively trap a novel conformational state of a protein kinase

Structural Characterization of the DEP Domains of P-Rex1

P-Rex1 is a guanine nucleotide exchange factor for Rho-GTPases, which is indirectly involved in the regulation of cell migration and proliferation. It contains a tandem DH/PH domain archetypal of the Dbl family of GEFs, two DEP and two PDZ domains, and a C-terminal end with weak homology to inositol polyphosphate 4-phosphatase. P-Rex1 is regulated by both intra-domain interactions and interactions with other proteins such as G-protein beta gamma, PKA and phosphatidylinositol (3,4,5)-trisphosphate. Upregulation of P-Rex1 has been found in multiple human cancers, making it a potential target for anti-cancer drug therapies. Therefore, structural characterization of P-Rex1 is critical. Currently, only the structures of the DH/PH tandem and PDZ1 domains of P-Rex1 have been determined. The goal of this project is to determine the structures of the DEP1 and DEP2 domains using X-Ray crystallography. P-Rex1-DEP1 (409-499 aa) protein was expressed in Escherichia coli and purified using affinity and size exclusion chromatography. The purified protein was then concentrated and used to set various crystallization screens. Small, well defined needles were observed and showed UV absorption, indicating that they consist of protein, and thus represent promising leads for a future structure determination. Optimization is in progress to grow bigger crystals or establish new conditions. Attempts are still being made to purify P-Rex1-DEP2 (500-602 aa), which thus far shows tendencies to aggregate

Identification of a G(iα) binding site on type V adenylyl cyclase

The stimulatory G protein α subunit G(sα) binds within a cleft in adenylyl cyclase formed by the α1-α2 and α3-β4 loops of the C2 domain. The pseudosymmetry of the C1 and C2 domains of adenylyl cyclase suggests that the homologous inhibitory α subunit G(iα) could bind to the analogous cleft within C1. We demonstrate that myristoylated guanosine 5\u27-3-O- (thio)triphosphate-G(iα1) forms a stable complex with the C1 (but not the C2) domain of type V adenylyl cyclase. Mutagenesis of the membrane-bound enzyme identified residues whose alteration either increased or substantially decreased the IC50 for inhibition by G(iα1). These mutations suggest binding of G(iα) within the cleft formed by the α2 and α3 helices of C1, analogous to the G(sα) binding site in C2. Adenylyl cyclase activity reconstituted by mixture of the C1 and C2 domains of type V adenylyl cyclase was also inhibited by G(iα). The C(1b) domain of the type V enzyme contributed to affinity for G(iα), but the source of C2 had little effect. Mutations in this soluble system faithfully reflected the phenotypes observed with the membrane-bound enzyme. The pseudosymmetrical structure of adenylyl cyclase permits bidirectional regulation of activity by homologous G protein α subunits

Crystal structure of the catalytic domains of adenylyl cyclase in a complex with G(sα)·GTPγΣ

The crystal structure of a soluble, catalytically active form of adenylyl cyclase in a complex with its stimulatory heterotrimeric G protein α subunit (G(sα)) and forskolin was determined to a resolution of 2.3 angstroms. When P-site inhibitors were soaked into native crystals of the complex, the active site of adenylyl cyclase was located and structural elements important for substrate recognition and catalysis were identified. On the basis of these and other structures, a molecular mechanism is proposed for the activation of adenylyl cyclase by G(sα)

Structure of RGS4 bound to AlF\u3csub\u3e4\u3c/sub\u3e\u3csup\u3e-\u3c/sup\u3e-activated G(iα1): Stabilization of the Transition State for GTP Hydrolysis

RGS proteins are GTPase activators for heterotrimeric G proteins. We report here the 2.8 Å resolution crystal structure of the RGS protein RGS4 complexed with G(iα1)-Mg2+-GDP-AlF4. Only the core domain of RGS4 is visible in the crystal. The core domain binds to the three switch regions of G(iα1), but does not contribute catalytic residues that directly interact with either GDP or AlF4. Therefore, RGS4 appears to catalyze rapid hydrolysis of GTP primarily by stabilizing the switch regions of G(iα1), although the conserved Asn-128 from RGS4 could also play a catalytic role by interacting with the hydrolytic water molecule or the side chain of Gln-204. The binding site for RGS4 on G(iα1) is also consistent with the activity of RGS proteins as antagonists of G(α) effectors

Crystal structure of the adenylyl cyclase activator G(sα)

The crystal structure of G(sα), the heterotrimeric G protein α subunit that stimulates adenylyl cyclase, was determined at 2.5 Å in a complex with guanosine 5\u27-O-(3-thio-triphosphate) (GTPγS). G(sα) is the prototypic member of a of GTP-binding proteins that regulate the activities of effectors in a hormone-dependent manner. Comparison of the structure of G(sα)·GTPγS with that of G(iα)·GTPγS suggest that their effector specificity is primarily dictated by the shape of the binding surface formed by the switch II helix and the α3-β5 loop, despite the high sequence homology of these elements. In contrast, sequence divergence explains the inability of regulators of G protein signaling to stimutate the GTPase activity of G(sα). The βγ binding surface ofG(sα) is largely conserved in sequence and structure to that of G(iα), whereas differences in the surface formed by the carboxyl-terminal helix and the α4-β6 loop may mediate ceptor specificity

Two-metal-ion catalysis in adenylyl cyclase

Adenylyl cyclase (AC) converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate, a ubiquitous second messenger that regulates many cellular functions. Recent structural studies have revealed much about the structure and function of mammalian AC but have not fully defined its active site or catalytic mechanism. Four crystal structures were determined of the catalytic domains of AC in complex with two different ATP analogs and various divalent metal ions. These structures provide a model for the enzyme- substrate complex and conclusively demonstrate that two metal ions bind in the active site. The similarity of the active site of AC to those of DNA polymerases suggests that the enzymes catalyze phosphoryl transfer by the same two-metal-ion mechanism and likely have evolved from a common ancestor

Synthesis of deuterium‐labelled amlexanox and its metabolic stability against mouse, rat, and human microsomes

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149374/1/jlcr3716_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149374/2/jlcr3716.pd

Structures of atypical chemokine receptor 3 reveal the basis for its promiscuity and signaling bias

Both CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are activated by the chemokine CXCL12 yet evoke distinct cellular responses. CXCR4 is a canonical G protein–coupled receptor (GPCR), whereas ACKR3 is intrinsically biased for arrestin. The molecular basis for this difference is not understood. Here, we describe cryo-EM structures of ACKR3 in complex with CXCL12, a more potent CXCL12 variant, and a small-molecule agonist. The bound chemokines adopt an unexpected pose relative to those established for CXCR4 and observed in other receptor-chemokine complexes. Along with functional studies, these structures provide insight into the ligand-binding promiscuity of ACKR3, why it fails to couple to G proteins, and its bias toward β-arrestin. The results lay the groundwork for understanding the physiological interplay of ACKR3 with other GPCRs