Solution structure and conformational heterogeneity of acylphosphatase from Bacillus subtilis

Abstract

a b s t r a c tAcylphosphatase is a small enzyme that catalyzes the hydrolysis of acyl phosphates. Here, we present the solution structure of acylphosphatase from Bacillus subtilis (BsAcP), the first from a Gram-positive bacterium. We found that its active site is disordered, whereas it converted to an ordered state upon ligand binding. The structure of BsAcP is sensitive to pH and it has multiple conformations in equilibrium at acidic pH (pH < 5.8). Only one main conformation could bind ligand, and the relative population of these states is modulated by ligand concentration. This study provides direct evidence for the role of ligand in conformational selection.

Ligand Binding Reduces Conformational Flexibility in the Active Site of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from Pseudomonas aeruginosa

“…1A). This lack of convergence in loop conformations has also been observed for other phosphatases, including the DUSP PRL-3 29 , a bacterial acylphosphatase 30 and DUSP PAC-1 31 .…”

“…Equally important, our studies also provide the first detailed description of functionally important loop dynamics in a DUSP in the absence of ligand and show how they change in response to ligand binding. Taken together, this work significantly extends the small but growing body of evidence suggesting cysteine-based phosphatases experience conformational flexibility in the open ligand-free state 29; 30; 31; 37; 38 . Furthermore, this is the first time that such an observation has been reported for a bacterial DUSP, showing that bacterial DUSPs may share not only structural but also dynamic similarities with their eukaryotic counterparts.…”

“…Moreover, even when open LMW-PTPs and DUSPs have been studied using NMR spectroscopy, NH cross-peaks for functionally relevant residues in the active loops are often missing. These observations suggest that these proteins are dynamic in the ligand-free open state, with the active site loops likely adopting multiple conformations 29; 30; 31; 37; 38 . Here, we show that for TpbA, addition of the simplest ligand, inorganic phosphate, is sufficient to induce a significant change in both the structure and dynamics of its active site loops, as evidenced by an increase of the number of NH crosspeaks in the ligand-bound 2D [ 1 H, 15 N] HSQC spectrum of TpbA.…”

“…Here, we show that for TpbA, addition of the simplest ligand, inorganic phosphate, is sufficient to induce a significant change in both the structure and dynamics of its active site loops, as evidenced by an increase of the number of NH crosspeaks in the ligand-bound 2D [ 1 H, 15 N] HSQC spectrum of TpbA. The phenomenon whereby ligand binding causes PTP loop resonances to appear in the NMR spectra has been observed in some phosphatases outside the DUSP family, such as LWM-PTPs 30; 37; 38 . However, the degree to which these active site loops experience dynamics, and, furthermore, on which timescales, have not yet been reported.…”

Protein Conformational Disorder and Enzyme Catalysis

“…Control experiments showed that enzymatic activity does not derive from the fraction of native protein in the sample, but substrate-induced organization of the active site could not be ruled out. NMR studies on a homologous Bacillus subtilis acylphosphatase indicated a structurally disordered active site in the absence of substrate that became more ordered upon substrate binding [84]. Conformational heterogeneity was particularly pronounced at acidic pH, where the enzyme exhibits optimal activity, possibly accounting for the relatively broad specificity of the enzyme.…”