Measuring Protein‐Protein Interactions by Equilibrium Sedimentation

Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2) 2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. Fatty acid kinase (Fak)2 is a two-protein system that consists of a dimeric ATP-binding protein (FakA) and a monomeric fatty acid-binding protein (FakB) (1). Fak expression is widespread in Gram-positive bacteria with most strains possessing a single FakA along with multiple FakBs. Staphylococcus aureus expresses two FakBs that both function with FakA. FakB1 binds a saturated fatty acid, although FakB2 prefers an unsaturated fatty acid (1). The FakBs possess a bound fatty acid, but they function as exchange proteins that swap a bound fatty acid for a fatty acid in a detergent micelle or membrane bilayer (Fig. 1). This exchange reaction underlies the function of Fak in exogenous fatty acid incorporation into membrane phospholipids in S. aureus (2). Extracellular fatty acids are flipped to the inner aspect of the membrane where they exchange onto a FakB. The acyl chain bound to FakB is phosphorylated by FakA, and the phosphorylated FakB then exchanges the acyl-PO 4 for a fatty acid in the membrane to initiate another round of phosphorylation. The deposited acyl-PO 4 is used by PlsY (glycerol-3-phosphate acyltransferase) to initiate membrane phospholipid synthesis or is converted to acyl-acyl carrier protein by PlsX (Fig. 1). Acyl-acyl carrier protein is used by either PlsC in the second acyltransferase step of phospholipid synthesis or by FabF, the elongation condensing enzyme of bacterial type II fatty acid synthesis (1). In addition to the role of FakA and FakB in the activation of exogenous fatt...

Section: Methodsmentioning

confidence: 99%

Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family

Broussard

Miller

Jackson

et al. 2016

“…Sedimentation equilibrium experiments were performed using Epon charcoal-filled six-sector centerpieces at the appropriate rpm with 0.001-cm spacing, scanned every 4 h, averaged from 10 replicates, and recorded at 545 nm to monitor the Cy3-labeled DNA. The data were processed and analyzed with SedFit/ SedPhat (Peter Schuck) (33)(34)(35)(36). The apparent molecular weights of the complexes were determined as described (30).…”

Section: Methodsmentioning

confidence: 99%

The Wrapping Loop and Rap1 C-terminal (RCT) Domain of Yeast Rap1 Modulate Access to Different DNA Binding Modes

Feldmann¹,

Bona

Galletto

2015

“…Absorbance was scanned at a wavelength interval of 0.001 cm with 20 replicates/step. The SE curves were analyzed using the non-linear regression analysis program SEDPHAT to obtain the K D , based on the Boltzmann distributions of ideal species in the centrifugal field (54). The SDS-PAGE and Western blotting assays under non-denaturing and denaturing conditions were used to evaluate protein integrity at the end of SV and SE experiments.…”

Section: Methodsmentioning

confidence: 99%

Structural Basis for the Binding Specificity of Human Recepteur d'Origine Nantais (RON) Receptor Tyrosine Kinase to Macrophage-stimulating Protein

Chao

Gorlatova

Eisenstein

et al. 2014

Background: RON and MET receptors bind their ligands MSP and HGF selectively and activate different signaling pathways. Results: Crystallographic and analytical ultracentrifugation studies provide important information about RON-MSP interaction. Conclusion: RON-MSP and MET-HGF exhibit 2:2 complex stoichiometry, but differences within the respective interfaces explain the strict ligand-receptor specificity. Significance: Signaling pathways must be exquisitely regulated with no cross-reactivity between related systems.