Single-crystal neutron diffraction is a powerful technique in structural biology that can accurately visualize the hydrogen atom positions in biomacromolecules. Because hydrogen atoms make up to 50% of all atoms in a protein, determine protonation states and facilitate enzyme function, pinpointing their locations contributes to an atomic-level understanding of proteins and invaluable information for structure-based drug design.
Tryptophan synthase (TS) is a pyridoxal 5’-phosphate (PLP, vitamin B6 derivative) -dependent enzyme found in plants, fungi, and bacteria but not in humans. TS is an attractive target for the design of inhibitors against pathogenic organisms such as Staphylococcus aureus and Mycobacterium tuberculosis. The goal of our study was to use neutron diffraction to shed new light on the TS catalytic mechanism by determining protonation states. TS is an αꞵꞵα heterotetramer with active sites catalyzing two individual reactions. In the α-active site, indole 3-glycerol-phosphate is cleaved into indole and glyceraldehyde-3-phosphate. Indole travels through an internal hydrophobic channel to the ꞵ-active site, where it reacts with a PLP-L-serine external aldimine to produce L-tryptophan. The most intriguing part of the mechanism involves a communication domain (ꞵ-comm) which senses indole in the channel to activate serine in the ꞵ-site.
TS was perdeuterated at the Deuteration Laboratory (D-Lab) in the Life Sciences Group at Institut Laue-Langevin (ILL, Grenoble, France) to increase the neutron scattering power of the crystals. To overcome the poor crystallization of TS in the lab, we carried out crystallization experiments on the International Space Station (ISS) using our Toledo Crystallization Box hardware [1]. Microgravity lacks the gravity-driven convection currents that may lead to irregular crystal feeding and depletion zones causing defects and hindering crystal growth.
Neutron-quality TS crystals grown in microgravity were used to perform data collection on the quasi-Laue neutron diffractometer (LADI-III) at the ILL. Thus, the all-atom structure of TS holoenzyme, with substrate-free α-subunit and covalently bound PLP internal aldimine state in the ꞵ-subunit, was obtained at 2.1 Å resolution [2].
Direct visualization of hydrogen atoms allowed protonation states to be assigned to the active site amino acid residues and the PLP cofactor. Structural analysis of the α-active site, assisted by pKa predictions, revealed how the catalytic glutamate residue may adopt two conformations with disparate pKa’s to obtain and donate a proton in the α-subunit reaction (Figure 1). This proton is involved in the cleavage of indole glycerol-3-phosphate, releasing indole whose presence in the channel leads to a conformational change in the α-subunit that relocates the ꞵ-comm domain signaling for the activation of serine by the ꞵ-subunit – a series of events initiated by a single proton. In the ꞵ-active site, we found the Schiff base nitrogen atom in the PLP internal aldimine was protonated, while the pyridine nitrogen and phenolic oxygen were both deprotonated (Figure 2). The observed protonation states depict how the protonation profile of PLP contributes to the reaction specificity and promotes ꞵ-elimination of the PLP-serine hydroxyl group to generate L-tryptophan. The knowledge gained from the neutron crystal structure of TS provides insight into the selective protonation of the PLP cofactor and electrostatic environments of the active sites that govern the enzyme’s catalytic function and inform future efforts to target TS for antibacterial therapeutics.
V.N. Drago (ORNL), M. Blakeley (ILL), J. Devos (ILL)
[1] Drago VN, Devos JM, Blakeley MP, Forsyth VT et al. (2022). NPJ Microgravity, 8, 13
[2] Drago VN, Devos JM, Blakeley MP, Forsyth VT et al. (2024) Cell Rep. Phys. Sci., 5, 101827
Figure 1. Dual conformations of α-active site residue Glu49. The 2|FO|-|FC| neutron scattering length density map is depicted in wheat mesh contoured at 1 σ. Hydrogen bond distances are given between the heteroatom and hydrogen atom. In the active position (Glu49a), Glu49 can donate a proton to indole 3-glycerol phosphate in the α-subunit reaction. Glu49 is oriented to receive a proton through a hydrogen bonding network in the Glu49b conformation.
Figure 2. Protonation states of TS b-subunit active site and PLP revealed by neutron diffraction. The 2|FO|-|FC| neutron scattering length density map is depicted in wheat mesh contoured at 1 σ, and the omit |FO|-|FC| neutron scattering length density is shown in purple mesh contoured at 2.2 σ. Hydrogen bond distances are given between the heteroatom and hydrogen atom. The Schiff base nitrogen, NSB, is protonated and makes and intramolecular hydrogen bond with the phenolic oxygen, O3′. Protonation of pyridine nitrogen, N1, is prevented by Ser377, which is stabilized by a hydrogen bond with Ser351. His86, positioned above the cofactor, is neutral and monoprotonated on the ε-nitrogen.
