Engineering Metamorphic Chemokine Lymphotactin/XCL1 into the GAG-Binding, HIV-Inhibitory Dimer Conformation.

Unlike other chemokines, XCL1 undergoes a distinct metamorphic interconversion between a canonical monomeric chemokine fold and a unique β-sandwich dimer. The monomeric conformation binds and activates the receptor XCR1, whereas the dimer binds extracellular matrix glycosaminoglycans and has been associated with anti-human immunodeficiency virus (HIV) activity. Functional studies of WT-XCL1 are complex, as both conformations are populated in solution.

Interconversion between two unrelated protein folds in the lymphotactin native state.

Proteins often have multiple functional states, which might not always be accommodated by a single fold. Lymphotactin (Ltn) adopts two distinct structures in equilibrium, one corresponding to the canonical chemokine fold consisting of a monomeric three-stranded beta-sheet and carboxyl-terminal helix. The second Ltn structure solved by NMR reveals a dimeric all-beta-sheet arrangement with no similarity to other known proteins.

An engineered second disulfide bond restricts lymphotactin/XCL1 to a chemokine-like conformation with XCR1 agonist activity.

Chemokines adopt a conserved tertiary structure stabilized by two disulfide bridges and direct the migration of leukocytes. Lymphotactin (Ltn) is a unique chemokine in that it contains only one disulfide and exhibits large-scale structural heterogeneity. Under physiological solution conditions (37 degrees C and 150 mM NaCl), Ltn is in equilibrium between the canonical chemokine fold (Ltn10) and a distinct four-stranded beta-sheet (Ltn40).

Evaluation of 3-hydroxy-3-methylglutaryl-coenzyme A lyase arginine-41 as a catalytic residue: use of acetyldithio-coenzyme A to monitor product enolization.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the divalent cation-dependent cleavage of HMG-CoA to produce acetyl-CoA and acetoacetate. Arginine-41 is an invariant residue in HMG-CoA lyases. Mutation of this residue (R41Q) correlates with human HMG-CoA lyase deficiency.

Investigation of conserved acidic residues in 3-hydroxy-3-methylglutaryl-CoA lyase: implications for human disease and for functional roles in a family of related proteins.

3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase catalyzes the divalent cation-dependent cleavage of HMG-CoA to form acetyl-CoA and acetoacetate. In metal-dependent aldol and Claisen reactions, acidic residues often function either as cation ligands or as participants in general acid/base catalysis. Site-directed mutagenesis was used to produce conservative substitutions for the conserved acidic residues Glu-37, Asp-42, Glu-72, Asp-204, Glu-279, and Asp-280.

Investigation of the oligomeric status of the peroxisomal isoform of human 3-hydroxy-3-methylglutaryl-CoA lyase.

Unprocessed 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, retaining the mitochondrial signal sequence, has been proposed to correspond to a peroxisomal isoform. Using a modified expression plasmid and purification protocol, it is now possible to isolate substantial amounts (>10mg) of highly purified peroxisomal HMG-CoA lyase. These improvements facilitate more detailed protein chemistry approaches for characterization of the enzyme, which exhibits substantial (eightfold) dithiothreitol (DTT) stimulation of activity.