Domain Movements upon Activation of Phenylalanine Hydroxylase Characterized by Crystallography and Chromatography-Coupled Small-Angle X-ray Scattering

Abstract

Mammalian phenylalanine hydroxylase (PheH) is an allosteric enzyme that catalyzes the first step in the catabolism of the amino acid phenylalanine. Following allosteric activation by high phenylalanine levels, the enzyme catalyzes the pterin-dependent conversion of phenylalanine to tyrosine. Inability to control elevated phenylalanine levels in the blood leads to increased risk of mental disabilities commonly associated with the inherited metabolic disorder, phenylketonuria. Although extensively studied, structural changes associated with allosteric activation in mammalian PheH have been elusive. Here, we examine the complex allosteric mechanisms of rat PheH using X-ray crystallography, isothermal titration calorimetry (ITC), and small-angle X-ray scattering (SAXS). We describe crystal structures of the pre-activated state of the PheH tetramer depicting the regulatory domains docked against the catalytic domains and preventing substrate binding. Using SAXS, we further describe the domain movements involved in allosteric activation of PheH in solution and present the first demonstration of chromatography-coupled SAXS with Evolving Factor Analysis (EFA), a powerful method for separating scattering components in a model-independent way. Together, these results support a model for allostery in PheH in which phenylalanine stabilizes the dimerization of the regulatory domains and exposes the active site for substrate binding and other structural changes needed for activity.

The regulatory domain of human tryptophan hydroxylase 1 forms a stable dimer

“…The present results predict that the quaternary structure of TPH1, and likely TPH2, resembles those of TyrH[25] and phenylalanine-activated PheH[10, 18]. Figure 4 shows a model for the structure of the dimer of the regulatory domain of tTPH1 based on these structures.…”

“…Key to this conformational change is formation of an ACT domain dimer by regulatory domains from two subunits[17, 18]. This model is supported by recent studies of the isolated regulatory domain[14, 19, 20] and of the intact protein[13, 18]. TyrH is activated by phosphorylation of Ser40 in its N-terminal regulatory domain and inhibited by catecholamines[10, 21].…”

“…Phenylalanine binding to an allosteric site in the regulatory domain[13, 14] causes a significant conformational change that opens up the active site[15, 16]. Key to this conformational change is formation of an ACT domain dimer by regulatory domains from two subunits[17, 18]. This model is supported by recent studies of the isolated regulatory domain[14, 19, 20] and of the intact protein[13, 18].…”

New protein structures provide an updated understanding of phenylketonuria

“…However, the equilibrium between RS-PAH and A-PAH depends upon Phe binding to another site on the protein, called the allosteric site, which is formed by a structural change repositioning various parts of the PAH protein in the transition from RS-PAH to A-PAH. The precise structural location of this allosteric site, which Fig 1 shows as present only in the A-PAH structure, was first hypothesized in 2013 (16); it is now strongly supported by newly published structural studies (17–20). In contrast, some earlier studies had generally suggested that allosteric Phe binding involved an intermolecular interaction involving the regulatory domain; these studies were interpreted solely on the basis of an RS-PAH structure model and did not foresee the conformational change required for formation of A-PAH (e.g.…”

Analysis of RNA structure using small-angle X-ray scattering

“…For conformational differences, the resulting SAXS data may still be used for ensemble modeling, but mass differences will require more careful purification or optimization of preparation protocols. A recently-described use of evolving factor analysis in preparation of SAXS samples by SEC may be useful for differentiating complex elution profiles [48]. …”