A novel approach to quantify G-protein-coupled receptor dimerization equilibrium using bioluminescence resonance energy transfer.

Along with other resonance energy transfer techniques, bioluminescence resonance energy transfer (BRET) has emerged as an important method for demonstrating protein-protein interactions in cells. In the field of G-protein-coupled receptors, including chemokine receptors, BRET has been widely used to investigate homo- and heterodimerization, a feature of their interactions that is emerging as integral to function and regulation. While demonstrating the existence of dimers for a given receptor proved to be fairly straightforward, quantitative comparisons of different receptors or mutants are nontrivial because of inevitable variations in the expression of receptor constructs.

The structure and dynamic properties of the complete histidine phosphotransfer domain of the chemotaxis specific histidine autokinase CheA from Thermotoga maritima.

The bacterial histidine autokinase CheA contains a histidine phosphotransfer (Hpt) domain that accepts a phosphate from the catalytic domain and donates the phosphate to either target response regulator protein, CheY or CheB. The Hpt domain forms a helix-bundle structure with a conserved four-helix bundle motif and a variable fifth helix. Observation of two nearly equally populated conformations in the crystal structure of a Hpt domain fragment of CheA from Thermotoga maritima containing only the first four helices suggests more mobility in a tightly packed helix bundle structure than previously thought.

A rapid and efficient way to obtain modified chemokines for functional and biophysical studies.

Chemokines and their receptors control cell migration associated with routine immune surveillance, inflammation and development. They are also implicated in a large number of inflammatory diseases, cancer and HIV. Here we describe a rapid and efficient way to express and purify milligram quantities of multiple chemokine ligands (CCL7/MCP-3, CCL14/HCC-1, CCL3/MIP-1α and CXCL8/IL-8) containing C-terminal modifications to enable coupling to fluorescent dyes or small molecules such as biotin, in vitro.

Emerging concepts and approaches for chemokine-receptor drug discovery.

Importance Of The Field: Chemokine receptors are most noted for their role in cell migration. However, inappropriate utilization or regulation of these receptors is implicated in many inflammatory diseases, cancer and HIV, making them important drug targets.

Areas Covered In This Review: Allostery, oligomerization and ligand bias are presented as they pertain to chemokine receptors and their associated pathologies.

Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists.

Chemokine receptors are critical regulators of cell migration in the context of immune surveillance, inflammation, and development. The G protein-coupled chemokine receptor CXCR4 is specifically implicated in cancer metastasis and HIV-1 infection. Here we report five independent crystal structures of CXCR4 bound to an antagonist small molecule IT1t and a cyclic peptide CVX15 at 2.

Structural basis for the localization of the chemotaxis phosphatase CheZ by CheAS.

CheA-short interacts with CheZ to localize CheZ to cell poles. The fifth helical region (residues 112 to 133) from the phosphotransfer domain of CheA interacts with CheZ and becomes ordered and helical, although it lacks a stable fold in the CheA fragment comprising residues 98 to 150 alone. One CheA molecule binds to one CheZ dimer.

Chapter 4. Interactions of chemokines with glycosaminoglycans.

Many proteins require interactions with cell surface glycosaminoglycans (GAGs) to exert their biologic activity. The effect of GAG binding on protein function ranges from essential roles in development, organogenesis, cell growth, cell adhesion, inflammation, tumorigenesis, and interactions with pathogens. A classic example is the role of GAGs in the interaction of fibroblast growth factors with their receptors, where GAGs play a role in specificity determination and control of receptor-ligand engagement.

Chapter 2. Homo- and hetero-oligomerization of chemokines.

Chemokines function in cell migration by binding and activating seven transmembrane G protein-coupled receptors (GPCRs) on leukocytes and many other diverse cell types. The extracellular binding event stabilizes specific conformations of the receptor that trigger cascades of intracellular signaling pathways involved in cell movement and activation (Baggiolini, 1998; Baggiolini et al., 1997; Charo and Ransohoff, 2006; Hartley et al.

Chemical-shift-perturbation mapping of the phosphotransfer and catalytic domain interaction in the histidine autokinase CheA from Thermotoga maritima.

Regulating the activity of the histidine autokinase CheA is a central step in bacterial chemotaxis. The CheA autophosphorylation reaction minimally involves two CheA domains, denoted P1 and P4. The kinase domain (P4) binds adenosine triphosphate (ATP) and orients the gamma phosphate for phosphotransfer to a reactive histidine on the phosphoacceptor domain (P1).

The contact interface of a 120 kD CheA-CheW complex by methyl TROSY interaction spectroscopy.

During bacterial chemotaxis, the histidine autokinase CheA interacts with the chemotaxis receptors with the help of the coupling protein CheW. This interaction is typical of many macromolecular complexes where protein-protein interactions play an important role. In this case, a relatively small protein, CheW, becomes part of a much larger complex.

Structural and chemical requirements for histidine phosphorylation by the chemotaxis kinase CheA.

The CheA histidine kinase initiates the signal transduction pathway of bacterial chemotaxis by autophosphorylating a conserved histidine on its phosphotransferase domain (P1). Site-directed mutations of neighboring conserved P1 residues (Glu-67, Lys-48, and His-64) show that a hydrogen-bonding network controls the reactivity of the phospho-accepting His (His-45) in Thermotoga maritima CheA. In particular, the conservative mutation E67Q dramatically reduces phosphotransfer to P1 without significantly affecting the affinity of P1 for the CheA ATP-binding domain.

Structural basis for the attachment of a paramyxoviral polymerase to its template.

The nucleocapsid of measles virus is the template for viral RNA synthesis and is generated through packaging of the genomic RNA by the nucleocapsid protein (N). The viral polymerase associates with the nucleocapsid through a small, trihelical binding domain at the carboxyl terminus of the phosphoprotein (P). Translocation of the polymerase along the nucleocapsid during RNA synthesis is thought to involve the repeated attachment and release of the binding domain.