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.

-Peptides differ from R-peptides by one additional backbone carbon atom and by resistance to metabolism and proteolysis. 1a-c -Peptides fold into helices, sheets, and turns and have wellrecognized potential as peptidomimetics. 2 Two -peptides that interact with discrete macromolecular targets are known: a -tetrapeptide hairpin that binds the somatostatin receptor with nanomolar affinity, 1d,e and a highly cationic 3 -decapeptide that binds TAR RNA. 1f Here, we report a set of 3 -peptides that possess significant 14-helix structure in water; one recognizes a cleft on the surface of the human oncogene product double minute 2 (hDM2) with nanomolar affinity.hDM2 is recognized in vivo by a short R-helix within the activation domain of p53 (p53AD, Figure 1A), the transcription factor that controls cell fate in response to stress. hDM2 negatively regulates p53 function, and disruption of the p53‚hDM2 interaction 3a is an important cancer therapy goal. 3b Three residues on one face of p53AD (F19, W23, and L26) comprise the functional epitope that contributes heavily to the binding energy. 3c-e Modification of a p53AD-based R-peptide with nonnatural R-amino acids that improve helix stability and surface complementarity results in a potent inhibitor that activates apoptosis in vivo. 3f,g The first small molecule inhibitors with submicromolar potency were reported in 2004. 3h Our design began with a 3 -decapeptide with significant 14-helix stability in aqueous solution due to electrostatic macrodipole stabilization and side chain-side chain salt bridges on one helical face. 4 Although the dimensions of a 14-helix differ from those of an R-helix, 2 we hypothesized that the p53AD functional epitope would be recapitulated if the side chains of F19, W23, and L26 were presented at successive positions three residues apart on a stabilized 14-helix ( Figure 1B). Because the 14-helix has almost exactly three residues per turn, these side chains should align upon folding. Four 3 -peptides were designed in which these side chains are appended in both possible orientations on each of the two available 14-helix faces ( 53-1-4, Figure 2).We compared the circular dichroism (CD) spectra of 53-1-4 in aqueous buffer to estimate their 14-helix content ( Figure 3A). 1g-j,2 The 14-helix signature is clearly evident, and the relative minima at 214 nm suggest helical contents between 30% and 50% for 53-1, 3, and 4. 5 Two-dimensional NMR spectroscopy 6 in CD 3 OH confirmed the presence of 14-helix structure in 53-1: ROESY spectra showed four of seven possible C R H(i)fC H(i+3) ROEs and two of six possible C N (i)fC (i+3) ROEs. Additional ROEs may be present but were obscured by resonance overlap; no ROEs inconsistent with 14-helical structure were observed. Analytical ultracentrifugation 5,6 revealed that 53-1, 3, and 4 were monomeric at concentrations between 80 and 400 µM, confirming that these 14-helices are stabilized by intramolecular interactions. We designed a competition fluorescence polarization (FP) assay 7 using hDM2 1-188 (hDM2...

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.

CXCL12 (SDF-1␣) and CXCR4 are critical for embryonic development and cellular migration in adults. These proteins are involved in HIV-1 infection, cancer metastasis, and WHIM disease. Sequestration and presentation of CXCL12 to CXCR4 by glycosaminoglycans (GAGs) is proposed to be important for receptor activation. Mutagenesis has identified CXCL12 residues that bind to heparin. However, the molecular details of this interaction have not yet been determined. Here we demonstrate that soluble heparin and heparan sulfate negatively affect CXCL12-mediated in vitro chemotaxis. We also show that a cluster of basic residues in the dimer interface is required for chemotaxis and is a target for inhibition by heparin. We present structural evidence for binding of an unsaturated heparin disaccharide to CXCL12 attained through solution NMR spectroscopy and x-ray crystallography. Increasing concentrations of the disaccharide altered the two-dimensional 1 H-15 N-HSQC spectra of CXCL12, which identified two clusters of residues. One cluster corresponds to ␤-strands in the dimer interface. The second includes the amino-terminal loop and the ␣-helix. In the x-ray structure two unsaturated disaccharides are present. One is in the dimer interface with direct contacts between residues His 25 , Lys 27 , and Arg 41 of CXCL12 and the heparin disaccharide. The second disaccharide contacts Ala 20 , Arg 21 , Asn 30 , and Lys 64 . This is the first x-ray structure of a CXC class chemokine in complex with glycosaminoglycans. Based on the observation of two heparin binding sites, we propose a mechanism in which GAGs bind around CXCL12 dimers as they sequester and present CXCL12 to CXCR4.