NMR solution structure and backbone dynamics of the CC chemokine eotaxin-3.

Eotaxin-3 is one of three related chemokines that specifically activate chemokine receptor CCR3. We report the 3D structure and backbone dynamics of eotaxin-3 determined by NMR spectroscopy. Eotaxin-3 is monomeric under the conditions in this study and consists of an unstructured N-terminus before the first two conserved cysteine residues, an irregularly structured N-loop following the second conserved cysteine, a single turn of 3(10)-helix, a three-stranded antiparallel beta-sheet, an alpha-helix, and an unstructured C-terminal tail. As in other chemokines, the alpha-helix packs against one face of the beta-sheet. The average backbone and heavy atom rmsd values of the 20 structures (residues 9-65) are 0.44 and 1.01 A, respectively. A comparison between the structures of eotaxin-3 and related chemokines suggests that the electrostatic potential in the vicinity of a surface groove and the structure of the beta2-beta3 turn may be important for maintaining receptor specificity. The backbone dynamics of eotaxin-3 were determined from 15N NMR relaxation data using the extended model free dynamics formalism. Large amplitude motions on the picosecond to nanosecond time scale were observed in both termini and in some residues in the N-loop, the beta1-beta2 turn, and the beta3 strand; the location of these residues suggests a possible role for dynamics in receptor binding and activation. In contrast to eotaxin, eotaxin-3 exhibits no substantial mobility on the microsecond to millisecond time scale.

Identification of receptor binding and activation determinants in the N-terminal and N-loop regions of the CC chemokine eotaxin.

Eotaxin is a CC chemokine that specifically activates the receptor CCR3 causing accumulation of eosinophils in allergic diseases and parasitic infections. Twelve amino acid residues in the N-terminal (residues 1-8) and N-loop (residues 11-20) regions of eotaxin have been individually mutated to alanine, and the ability of the mutants to bind and activate CCR3 has been determined in cell-based assays. The alanine mutants at positions Thr(7), Asn(12), Leu(13), and Leu(20) show near wild type binding affinity and activity. The mutants T8A, N15A, and K17A have near wild type binding affinity for CCR3 but reduced receptor activation. A third class of mutants, S4A, V5A, R16A, and I18A, display significantly perturbed binding affinity for CCR3 while retaining the ability to activate or partially activate the receptor. Finally, the mutant Phe(11) has little detectable activity and 20-fold reduced binding affinity relative to wild type eotaxin, the most dramatic effect observed in both assays but less dramatic than the effect of mutating the corresponding residue in some other chemokines. Taken together, the results indicate that residues contributing to receptor binding affinity and those required for triggering receptor activation are distributed throughout the N-terminal and N-loop regions. This conclusion is in contrast to the separation of binding and activation functions between N-loop and N-terminal regions, respectively, that has been observed previously for some other chemokines.

A test of the relationship between sequence and structure in proteins: excision of the heme binding site in apocytochrome b5.

The water-soluble domain of rat hepatic holocytochrome b5 is an alphabeta protein containing elements of secondary structure in the sequence beta1-alpha1-beta4-beta3-alpha2-alpha3-beta5- alpha4-alpha5-beta2-alpha6. The heme group is enclosed by four helices, a2, a3, a4, and a5. To test the hypothesis that a small b hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to beta1-alpha1-beta4-beta3-lambda-beta2-alpha6 was prepared, where lambda is a seven-residue linker bypassing the heme binding site. The fragment ("abridged b5") was found to contain alpha and beta secondary structure by circular dichroism spectroscopy and tertiary structure by Trp fluorescence emission spectroscopy. NMR data revealed a species with spectral properties similar to those of the full-length apoprotein. This folded form is in slow equilibrium on the chemical shift time scale with other less folded species. Thermal denaturation, as monitored by circular dichroism, absorption, and fluorescence spectroscopy, as well as size-exclusion chromatography-fast protein liquid chromatography (SEC-FPLC), confirmed the coexistence of at least two distinct conformational ensembles. It was concluded that the protein fragment is capable of adopting a specific fold likely related to that of cytochrome b5, but does not achieve high thermodynamic stability and cooperativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the dominating influence to restrict the conformational space searched by the chain is structural propensities at a local level rather than internal packing. The sequence also holds the properties necessary to generate a barrier to unfolding.

Design challenges for hemoproteins: the solution structure of apocytochrome b5.

In order to characterize the structural and dynamic factors that determine the assembly in b hemoproteins, the solution structure of the 98-residue protein apocytochrome b5 was determined by NMR methods. Over 800 experimental restraints derived from a series of two- and three-dimensional experiments were used. Holocytochrome b5, the protein with iron protoporphyrin-IX liganded to His-39 and His-63, contains in sequence the following elements of secondary structure: beta 1-alpha 1-beta 4-beta 3-alpha 2-alpha 3-beta 5-alpha 4-alpha 5-beta 2-alpha 6 [Mathews, F.S., Czerwinski, E. W., & Argos, P. (1979) The Porphyrins, Vol. 7, pp. 107-147, Academic Press, New York]. The folded holoprotein possesses two hydrophobic cores: an extensive, functional core around the heme (core 1), and a smaller, structural core remote from the heme (core 2). The apoprotein was found to contain a stable four-stranded beta-sheet encompassing beta 1, beta 2, beta 3, and beta 4 and three alpha-helices, corresponding to alpha 1, alpha 2, and alpha 6. Two short alpha-helices (alpha 3 and alpha 5) appear to form partially, and alpha 4 is not detected. These three helices and beta 5 border the heme binding pocket and are disordered in the apoprotein NMR structure. According to backbone 1H-15N NOE results, the most flexible region of the apoprotein, except for the termini, extends from Ala-50 (in beta 5) to Glu-69 (in alpha 5). The polypeptide segment bearing His-63 (located immediately prior to alpha 5) exhibits faster internal motions than that bearing His-39 (at the C-terminal end of alpha 2). The latter imidazole samples a restricted region of space, whereas the former can adopt many orientations with respect to the stable core. It was concluded that heme removal affects the structure and dynamics of most of core 1 whereas it leaves core 2 largely intact. The results provide guidelines for the rational design of b hemoproteins: a modular structure including a packed, stable core and a partially folded binding site is anticipated to present strong kinetic and thermodynamic advantages compared to approaches relying on the complete formation of secondary structure prior to heme binding.