PDBsum entry 1b50

Chemokine

PDB id: 1b50

Contents

Protein chains

69 a.a. *

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Identification of amino acid residues critical for aggregation of human cc chemokines macrophage inflammatory protein (mip)-1alpha, Mip-1beta, And rantes. Characterization of active disaggregated chemokine variants.

Authors

L.G.Czaplewski, J.Mckeating, C.J.Craven, L.D.Higgins, V.Appay, A.Brown, T.Dudgeon, L.A.Howard, T.Meyers, J.Owen, S.R.Palan, P.Tan, G.Wilson, N.R.Woods, C.M.Heyworth, B.I.Lord, D.Brotherton, R.Christison, S.Craig, S.Cribbes, R.M.Edwards, S.J.Evans, R.Gilbert, P.Morgan, E.Randle, N.Schofield, P.G.Varley, J.Fisher, J.P.Waltho, M.G.Hunter.

Ref.

J Biol Chem, 1999, 274, 16077-16084. [DOI no: 10.1074/jbc.274.23.16077]

PubMed id

10347159

Abstract

Human CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation normal T cell expressed) self-associate to form high-molecular mass aggregates. To explore the biological significance of chemokine aggregation, nonaggregating variants were sought. The phenotypes of 105 hMIP-1alpha variants generated by systematic mutagenesis and expression in yeast were determined. hMIP-1alpha residues Asp26 and Glu66 were critical to the self-association process. Substitution at either residue resulted in the formation of essentially homogenous tetramers at 0.5 mg/ml. Substitution of identical or analogous residues in homologous positions in both hMIP-1beta and RANTES demonstrated that they were also critical to aggregation. Our analysis suggests that a single charged residue at either position 26 or 66 is insufficient to support extensive aggregation and that two charged residues must be present. Solution of the three-dimensional NMR structure of hMIP-1alpha has enabled comparison of these residues in hMIP-1beta and RANTES. Aggregated and disaggregated forms of hMIP-1alpha, hMIP-1beta, and RANTES generally have equivalent G-protein-coupled receptor-mediated biological potencies. We have therefore generated novel reagents to evaluate the role of hMIP-1alpha, hMIP-1beta, and RANTES aggregation in vitro and in vivo. The disaggregated chemokines retained their human immunodeficiency virus (HIV) inhibitory activities. Surprisingly, high concentrations of RANTES, but not disaggregated RANTES variants, enhanced infection of cells by both M- and T-tropic HIV isolates/strains. This observation has important implications for potential therapeutic uses of chemokines implying that disaggregated forms may be necessary for safe clinical investigation.

Figure 1.
Fig. 1. Identification of fully active disaggregated mutants of hMIP-1 . A, the number of single amino acid substitutions generated at each residue in hMIP-1 . For a full description of all substitutions see Craig et al. (41). The number of alternative amino acid substitutions at each residue, which expressed well ( 20% hMIP-1 ), is shown above the origin and those which expressed poorly ( 20% hMIP-1 ) below the origin. Cysteine residues at amino acid positions 10, 11, 34, and 50 were not mutated to retain structural integrity. B and subsequent panels refer to amino acid residues at which the described properties have been identified are shown with a tall histogram and variants which were assayed for a property but did not meet the criteria (e.g. they were not disaggregated or they were less potent) are shown by short histograms. B, variants that expressed well and were disaggregated according to native polyacrylamide gel electrophoresis. Disaggregated variants migrated substantially further into the gel than hMIP-1 , which remained near the well. C, variants that were disaggregated according to sedimentation equilibrium AUC analysis. Disaggregated variants possessed weight average molecular weights 100,000 Da. D, disaggregated variants that retained full competitive receptor binding activity on FDCP-mix A4 cells. Fully active variants were defined as those with IC[50] values 8 nM in this cellular assay.

Figure 4.
Fig. 4. Three-dimensional structure of hMIP-1 D26A and comparative positions of amino acid residues in hMIP-1 , hMIP-1 , and RANTES . A, stereo view of the overlay of the monomer backbone structures of hMIP-1 D26A and hMIP-1 . The regions 1-15, 31-38, and 67-69, which are discussed in the text, are shown as a thin trace, whereas the rest of the backbone is shown as a thick trace. B, from left to right, space-filling representations of the three-dimensional NMR structures of hMIP-1 D26A, RANTES (6), and hMIP-1 (5) are shown to illustrate the relative positions of the key acidic amino acid residues involved in chemokine self-association at positions 26 and 66 (27 and 67 in hMIP-1 ) shaded in dark gray. The positions of the basic residues in the 44, 45, and 47 positions (positions 45, 46, and 47 in hMIP-1 ), which we speculate may be involved in charge interactions leading to self-association are shaded in light gray. The terminal residues 1-15 and 67-69 are not shown in this figure.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (1999, 274, 16077-16084) copyright 1999.