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MIP-2/CXCL2 is a murine chemokine related to human chemokines that possesses the Glu-Leu-Arg (ELR) activation motif and activates CXCR2 for neutrophil chemotaxis. We determined the structure of MIP-2 to 1.9 Å resolution and created a model with its murine receptor CXCR2 based on the coordinates of human CXCR4. Chemokine-induced migration of cells through specific G-protein coupled receptors is regulated by glycosaminoglycans (GAGs) that oligomerize chemokines. MIP-2 GAG-binding residues were identified that interact with heparin disaccharide I-S by NMR spectroscopy. A model GAG/MIP-2/CXCR2 complex that supports a 2:2 complex between chemokine and receptor was created. Mutants of these disaccharide-binding residues were made and tested for heparin binding, in vitro neutrophil chemotaxis, and in vivo neutrophil recruitment to the mouse peritoneum and lung. The mutants have a 10-fold decrease in neutrophil chemotaxis in vitro. There is no difference in neutrophil recruitment between wild-type MIP-2 and mutants in the peritoneum, but all activity of the mutants is lost in the lung, supporting the concept that GAG regulation of chemokines is tissue-dependent.

The structural and functional properties of human prolactin (hPRL), a 23 kDa protein hormone and cytokine, are pH-dependent. The dissociation rate constant for binding to the extracellular domain of the hPRL receptor increases nearly 500-fold over the relatively narrow and physiologic range from pH 8 to 6. As the apparent midpoint for this transition occurs around pH 6.5, we have looked toward histidine residues as a potential biophysical origin of the behavior. hPRL has a surprising number of nine histidines, nearly all of which are present on the protein surface. Using NMR spectroscopy, we have monitored site-specific proton binding to eight of these nine residues and derived equilibrium dissociation constants. During this analysis, a thermodynamic interaction between a localized triplet of three histidines (H27, H30, and H180) became apparent, which was subsequently confirmed by site-directed mutagenesis. After consideration of multiple potential models, we present statistical support for the existence of two negative cooperativity constants, one linking protonation of residues H30 and H180 with a magnitude of approximately 0.1 and the other weaker interaction between residues H27 and H30. Additionally, mutation of any of these three histidines to alanine stabilizes the folded protein relative to the chemically denatured state. A detailed understanding of these complex protonation reactions will aid in elucidating the biophysical mechanism of pH-dependent regulation of hPRL's structural and functional properties.

A member of the family of hematopoietic cytokines, human prolactin (hPRL) serves a dual role both as an endocrine hormone and as an autocrine/paracrine cytokine or growth factor. During investigation of the solution structural properties of hPRL, we have noted a surprising pH dependence of its structural stability over a range from approximately pH 6.0 to pH 8.0. An analysis of backbone atom NMR chemical shift changes and backbone amide hydrogen-deuterium exchange rates due to titration of the solution pH over this same range, along with calculations of protein surface electrostatic potential, suggests the possible involvement of a localized cluster of three His residues (27, 30, and 180), which comprise a portion of the high-affinity receptor-binding epitope. Surface plasmon resonance analysis of the interaction between hPRL and the extracellular domain (ECD) of the hPRL receptor reveals a selective 500-fold change in the dissociation rate between pH 8.3 and pH 5.8. In comparison, the interaction of hGH with the same receptor ECD did not demonstrate any significant dependence on pH. We also present an initial investigation of the pH dependence of hPRL function in rat Nb2 cell proliferation assays and a STAT5 luciferase gene reporter assay in the T47D human breast cancer cell line, whose results are consistent with our biophysical studies. The potential implications of this variation in hPRL's structural stability and receptor-binding kinetics over this physiologic range of pH are discussed.

In humans, the enzyme thiopurine methyltransferase (TPMT) metabolizes 6-thiopurine (6-TP) medications, commonly used for immune suppression and for the treatment of hematopoietic malignancies. Genetic polymorphisms in the TPMT protein sequence accelerate intracellular degradation of the enzyme through an ubiquitylation and proteasomal-dependent pathway. Research has led to the hypothesis that these polymorphisms destabilize the native structure of TPMT, resulting in the formation of misfolded or partially unfolded states, which are subsequently recognized for intracellular degradation. Addition of the cosubstrate, S-adenosylmethionine (SAM), prevents degradation of the TPMT polymorphs in experimental assays, presumably by stabilizing the native structure. Using a bacterial orthologue of TPMT from Pseudomonas syringae, we have used NMR spectroscopy to describe the consequences of binding sinefungin, a SAM analogue, on the structure and dynamics of the TPMT protein backbone. NMR chemical shift mapping experiments localize sinefungin to a highly conserved site in classical methyltransferases. Distal chemical shift changes involving the presumed active site cover imply indirect conformational changes induced by sinefungin, which may play a role in substrate recognition or the catalytic mechanism. Analysis of protein backbone dynamics based on NMR relaxation reveals a combination of complementary effects. Whereas the peripheral, inserted structural elements of the TPMT topology are conformationally stabilized by the presence of sinefungin, a consistent increase in backbone mobility is observed for the central, conserved structural elements. The potential implications for the structural and dynamic effects of binding sinefungin for the catalytic mechanism of the enzyme and the stabilization of the degradation-susceptible TPMT polymorphs are discussed.

The intestinal fatty acid binding protein is composed of two beta-sheets surrounding a large interior cavity. There is a small helical domain associated with the portal for entry of the ligand into the cavity. Denaturation of the protein has been monitored in a residue-specific manner by collecting a series of two-dimensional (1)H-(15)N heteronuclear single-quantum coherence (HSQC) NMR spectra from 0 to 6.5 M urea under equilibrium conditions. In addition, rates for hydrogen-deuterium exchange have been measured as a function of denaturant concentration. Residual, native-like structure persists around hydrophobic clusters at very high urea concentrations. This residual structure (reflecting only about 2-7% persistence of native-like structure) involves the turns between beta-strands and between the two short helices. If this persistence is assumed to reflect transient native-like structure in these regions of the polypeptide chain, these sites may serve as nucleation sites for folding. The data obtained at different urea concentrations are then analyzed on the basis of peak intensities relative to the intensities in the absence of urea reflecting the extent of secondary structure formation. At urea concentrations somewhat below 6.5 M, specific hydrophobic residues in the C-terminal beta-sheet interact and two strands, the D and E strands in the N-terminal beta-sheet, are stabilized. These latter strands surround one of the turns showing residual structure. With decreasing urea concentrations, the remaining strands are stabilized in a specific order. The early strand stabilization appears to trigger the formation of the remainder of the C-terminal beta-sheet. At low urea concentrations, hydrogen bonds are formed. A pathway is proposed on the basis of the data describing the early, intermediate, and late folding steps for this almost all beta-sheet protein. The data also show that there are regions of the protein which appear to act in a concerted manner at intermediate steps in refolding.

The backbone dynamics of the liganded (holo) and unliganded (apo) forms of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) have been characterized and compared using amide 15N relaxation and 1H exchange NMR measurements. The amide 1H/15N resonances for apo and holo I-FABP were assigned at 25 degrees C, and gradient- and sensitivity-enhanced 2D experiments were employed to measure l5N T1, T2, and [1H]15N NOE values and relative 1H saturation transfer rates. The 15N relaxation parameters were analyzed using five different representations of the spectral density function based on the Lipari and Szabo formalism. A majority of the residues in both apo and holo I-FABP were characterized by relatively slow hydrogen exchange rates, high generalized order parameters, and no conformational exchange terms. However, residues V26-N35, S53-R56, and A73-T76 of apo I-FABP were characterized by rapid hydrogen exchange, low order parameters, and significant conformational exchange. These residues are clustered in a single region of the protein where variability and apparent disorder were previously observed in the chemical shift analyses and in the NOE-derived NMR structures of apo I-FABP. The increased mobility and discrete disorder in the backbone of the apo protein may permit the entry of ligand into the binding cavity. We postulate that the bound fatty acid participates in a series of long-range cooperative interactions that cap and stabilize the C-terminal half of helix II and lead to an ordering of the portal region. This ligand-modulated order-disorder transition has implications for the role of I-FABP in cellular fatty acid transport and targeting.

The three-dimensional structure of the unliganded form of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) has been determined using triple-resonance three-dimensional nuclear magnetic resonance (3D NMR) methods. Sequence-specific 1H, 13C, and 15N resonance assignments were established at pH 7.2 and 33 degrees C and used to determine the consensus 1H/13C chemical shift-derived secondary structure. Subsequently, an eight-stage iterative procedure was used to assign the 3D 13C- and 15N-resolved NOESY spectra, yielding a total of 3335 interproton distance restraints or 26 restraints/residue. The tertiary structures were calculated using a distance geometry/simulated annealing algorithm that employs pairwise Gaussian metrization to achieve improved sampling and convergence. The final ensemble of NMR structures exhibited a backbone conformation generally consistent with the beta-clam motif described for members of the lipid-binding protein family. However, unlike holo-I-FABP, the structure ensemble for apo-I-FABP exhibited variability in a discrete region of the backbone. This variability was evaluated by comparing the apo- and holoproteins with respect to their backbone 1H and 13C chemical shifts, amide 1H exchange rates, and 15N relaxation rates. Together, these results established that the structural variability represented backbone disorder in apo-I-FABP. The disorder was most pronounced in residues K29-L36 and N54-N57, encompassing the distal half of alpha-helix II, the linker between helix II and beta-strand B, and the reverse turn between beta-strands C and D. It was characterized by a destablization of long-range interactions between helix II and the C-D turn and a fraying of the C-terminal half of the helix. Unlike the solution-state NMR structure, the 1.2-A X-ray crystal structure of apo-I-FABP did not exhibit this backbone disorder. In solution, the disordered region may function as a dynamic portal that regulates the entry and exit of fatty acid. We hypothesize that fatty acid binding shifts the order-disorder equilibrium toward the ordered state and closes the portal by stabilizing a series of cooperative interactions resembling a helix capping box. This proposed mechanism has implications for the acquisition, release, and targeting of fatty acids by I-FABP within the cell.