Structural Basis for Allosteric Ligand Recognition in the Human CC Chemokine Receptor 7.

The CC chemokine receptor 7 (CCR7) balances immunity and tolerance by homeostatic trafficking of immune cells. In cancer, CCR7-mediated trafficking leads to lymph node metastasis, suggesting the receptor as a promising therapeutic target. Here, we present the crystal structure of human CCR7 fused to the protein Sialidase NanA by using data up to 2.1 Å resolution. The structure shows the ligand Cmp2105 bound to an intracellular allosteric binding pocket. A sulfonamide group, characteristic for various chemokine receptor ligands, binds to a patch of conserved residues in the Gi protein binding region between transmembrane helix 7 and helix 8. We demonstrate how structural data can be used in combination with a compound repository and automated thermal stability screening to identify and modulate allosteric chemokine receptor antagonists. We detect both novel (CS-1 and CS-2) and clinically relevant (CXCR1-CXCR2 phase-II antagonist Navarixin) CCR7 modulators with implications for multi-target strategies against cancer.

Quadruple space-group ambiguity owing to rotational and translational noncrystallographic symmetry in human liver fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphatase (FBPase) is a key regulator of gluconeogenesis and a potential drug target for type 2 diabetes. FBPase is a homotetramer of 222 symmetry with a major and a minor dimer interface. The dimers connected via the minor interface can rotate with respect to each other, leading to the inactive T-state and active R-state conformations of FBPase. Here, the first crystal structure of human liver FBPase in the R-state conformation is presented, determined at a resolution of 2.2 Šin a tetragonal setting that exhibits an unusual arrangement of noncrystallographic symmetry (NCS) elements. Self-Patterson function analysis and various intensity statistics revealed the presence of pseudo-translation and the absence of twinning. The space group is P41212, but structure determination was also possible in space groups P43212, P4122 and P4322. All solutions have the same arrangement of three C2-symmetric dimers spaced by 1/3 along an NCS axis parallel to the c axis located at (1/4, 1/4, z), which is therefore invisible in a self-rotation function analysis. The solutions in the four space groups are related to one another and emulate a body-centred lattice. If all NCS elements were crystallographic, the space group would be I4122 with a c axis three times shorter and a single FBPase subunit in the asymmetric unit. I4122 is a minimal, non-isomorphic supergroup of the four primitive tetragonal space groups, explaining the space-group ambiguity for this crystal.

Determination of protein-ligand binding constants of a cooperatively regulated tetrameric enzyme using electrospray mass spectrometry.

This study highlights the benefits of nano electrospray ionization mass spectrometry (nanoESI-MS) as a fast and label-free method not only for determination of dissociation constants (KD) of a cooperatively regulated enzyme but also to better understand the mechanism of enzymatic cooperativity of multimeric proteins. We present an approach to investigate the allosteric mechanism in the binding of inhibitors to the homotetrameric enzyme fructose 1,6-bisphosphatase (FBPase), a potential therapeutic target for glucose control in type 2 diabetes. A series of inhibitors binding at an allosteric site of FBPase were investigated to determine their KDs by nanoESI-MS. The KDs determined by ESI-MS correlate very well with IC50 values in solution. The Hill coefficients derived from nanoESI-MS suggest positive cooperativity. From single-point measurements we could obtain information on relative potency, stoichiometry, conformational changes, and mechanism of cooperativity. A new X-ray crystal structure of FBPase tetramer binding ligand 3 in a 4:4 stoichiometry is also reported. NanoESI-MS-based results match the current understanding of the investigated system and are in agreement with the X-ray structural data, but provide additional mechanistic insight on the ligand binding, due to the better dynamic resolution. This method offers a powerful approach for studying other proteins with allosteric binding sites, as well.

Structure of the acid-sensing ion channel 1 in complex with the gating modifier Psalmotoxin 1.

Venom-derived peptide toxins can modify the gating characteristics of excitatory channels in neurons. How they bind and interfere with the flow of ions without directly blocking the ion permeation pathway remains elusive. Here we report the crystal structure of the trimeric chicken Acid-sensing ion channel 1 in complex with the highly selective gating modifier Psalmotoxin 1 at 3.0 Å resolution. The structure reveals the molecular interactions of three toxin molecules binding at the proton-sensitive acidic pockets of Acid-sensing ion channel 1 and electron density consistent with a cation trapped in the central vestibule above the ion pathway. A hydrophobic patch and a basic cluster are the key structural elements of Psalmotoxin 1 binding, locking two separate regulatory regions in their relative, desensitized-like arrangement. Our results provide a general concept for gating modifier toxin binding suggesting that both surface motifs are required to modify the gating characteristics of an ion channel.

Hydrophilic interaction chromatography of intact, soluble proteins.

The separation of intact proteins by means of Hydrophilic Interaction Chromatography (HILIC) was demonstrated with human apoA-I, recombinant human apoM, and equine cytochrome C. Five different commercially available HILIC columns were compared. Using one of these columns, different glycosylated isoforms of apoM were separated from each other and from the aglyco-form.