Cysteine-reactive Probes Research Articles

Binding-Site Chemical Probing in Homology Models Using Affinity Labeling of Cysteine-Substituted Receptors

The accuracy of the docking of a ligand in its modeled binding-site depends on the reliability of this model. To provide a model with experimental support, we have developed an engineered affinity labeling method combining cysteine-reactive probes with substitutions of putative site-lining residues into cysteines. This strategy amounts to building chemical sensors for the proximity of the substituted cysteines; it requires an activity or binding assay to monitor the irreversible occupancy of the site by the reactive ligand. Using affinity probes made reactive in different positions, the docking of the ligand can be inferred from the observed pattern of coupling reactions. The method involves three steps: ligand chemistry, mutagenesis and biological assays, which are detailed and scrutinized in the review: lead selection, ligand derivatization, and evaluation of the affinity probes (stability, reactivity and biological properties) for the ligands; positional selection and mutant properties for the cysteine substitutions; functional controls and assays for the analysis of the irreversible reactions. Examples illustrate the different criteria of concern; the data are interpreted in terms of binding-site structure and function. Potentially, the method can explore protein dynamics, since its targets are full-length membrane-inserted heteromeric proteins: it can detect subtype-dependent or activation-induced conformational changes.

An actin-dependent conformational change in myosin.

Conformational changes within myosin lead to its movement relative to an actin filament. Several crystal structures exist for myosin bound to various nucleotides, but none with bound actin. Therefore, the effect of actin on the structure of myosin is poorly understood. Here we show that the swing of smooth muscle myosin lever arm requires both ADP and actin. This is the first direct observation that a conformation of myosin is dependent on actin. Conformational changes within myosin were monitored using fluorescence resonance energy transfer techniques. A cysteine-reactive probe is site-specifically labeled on a 'cysteine-light' myosin variant, in which the native reactive cysteines were removed and a cysteine engineered at a desired position. Using this construct, we show that the actin-dependent ADP swing causes an 18 A change in distance between a probe on the 25/50 kDa loop on the catalytic domain and a probe on the regulatory light chain, corresponding to a 23 degrees swing of the light-chain domain.

Glutamate 779, an intramembrane carboxyl, is essential for monovalent cation binding by the Na,K-ATPase.

Incubation of purified renal Na,K-ATPase with the fluorescent carboxyl-selective reagent, 4-(diazomethyl)-7-(diethylamino)- coumarin (DEAC), results in enzyme inactivation via disruption of the monovalent cation binding sites and loss of K+ and Na+ binding capacity. Modification of 1 or 2 carboxyl residues in the alpha-subunit in a K+ or Na(+)-preventable manner leaves the ATP binding unaltered, and the enzyme is still able to undergo the major conformational transitions (Argüello, J. M., and Kaplan, J. H. (1991) J. Biol. Chem. 266, 14627-14635). Labeled alpha-subunits of Na,K-ATPase were isolated by gel electrophoresis and digested using V8 protease. The digests contained two bands which were fluorescently labeled in a cation-protectable fashion. Four peptides were identified in these bands. Peptides I (apparent molecular mass approximately 17 kDa), II (approximately 15 kDa), and IIIa (approximately 5 kDa) start at Gly758 while Peptide IIIb (approximately 5 kDa) starts at Gly561. Subsequent proteolysis of peptides IIIa and IIIb with thermolysin followed by electrophoresis revealed a single smaller fluorescent peptide which passed through 3-kDa cut-off membrane filters but was retained by 1-kDa cut off filters. N-terminal sequence analysis of this peptide gave the sequence 773Leu-Thr-Ser-Asn-Ile-Pro-Glu-Ile-Thr-Pro-Phe-Leu. The length of this peptide was also examined in labeling experiments with cysteine-reactive probes which indicated that the peptide did not extend to the next carboxyl-containing amino acid residue in the alpha-subunit sequence (Asp804). The site of attachment of DEAC is thus Glu779, an intramembrane carboxyl residue present in all known sequences of alpha-subunit isoforms of the Na,K-ATPase. This glutamate is essential for Na+ and K+ binding and active transport by the sodium pump. Its location in the fifth transmembrane segment provides a way in which information about ATP binding and phosphorylation in the major cytoplasmic loop of the enzyme is transmitted to intramembrane cation sites during the reaction cycle.