The pH Effect on Backbone Dynamics of the Antifreeze‐like Domain of Human Sialic Acid Synthase

Sialic acids (N-acetylneuraminic acids) play important roles in various biological functions, such as transmembrane signaling, cell growth, and cell adhesion. In bacteria, sialic acids are synthesized by a conserved enzymatic pathway including sialic acid synthase (SAS) that converts phosphoenolpyruvate (PEP) andN-acetylmannosamine (ManNAc) into N-acetylneuraminic acid (NeuNAc, or sialic acid). Mammalian SAS catalyzes the condensation of PEP and ManNAc 6-phosphate into NeuNAc 9-phosphate which is dephosphorylated into sialic acid. The crystal structure of an SAS (nmSAS) from Neisseria meningitides, in complex with Mn, PEP, andManNAc, revealed a unique domain-swapped homodimer consisting of an N-terminal catalytic NeuB domain of (α/β)8 barrel-type fold and a C-terminal antifreeze-like (AFL) domain with high structural similarity to the type III antifreeze proteins (AFPs). A nuclear magnetic resonance (NMR) study of the human AFL (hAFL) domain found that the structurewas similar to those of the type IIIAFPs. In nmSAS, the highly conversedR314 (Figure 1(a)) in the AFL domain (nmAFL) forms direct and watermediated hydrogen bonding (H-bonding) interaction with the acetyl group oxygen of ManNAc. It was proposed that the deletion of the AFL domain in human SAS (hAFL) would abolish the SAS activity, indicating that the hAFL domain should play an important role in substrate binding by the SAS. Therefore, the AFL domain is thought to play crucial roles in substrate binding of theNeuBdomain of the opposite monomer. In this study, we have investigated backbone dynamics of the hAFL domain at two different pHs. We also characterized structural and dynamic properties of hAFL by analyzing the temperature gradient of the amide proton chemical shifts. The results revealed that most residues of hAFL are relatively rigid at the higher temperature while the substrate-binding residues exhibit unusual flexible backbone dynamics at the lower temperature. The study provides insight into the pH dependency of backbone dynamics of hAFL and its substrate binding. The NMR chemical shift is sensitive to temperature-related structural or dynamic changes. As temperature was lowered, significant movements of cross-peaks were observed for most residues in the H/N heteronuclear single quantum correlation (HSQC) spectra of hAFL. Figure 1(b) shows the temperature gradients of amide proton chemical shifts (Δδ/ΔT) at pH = 6.0 and 8.0 as a function of residue numbers. Several residues haveΔδ/ΔT values > −5.0 ppb/K, indicating that these amides areH-bonded.ResidueswithΔδ/ΔTvalues < −5.0 ppb/K are not protected from solvent exchange by Hbonding, which is in good agreement with the NMR structure of hAFL. Residues G304, D310, G317, I327, G332, V335, and E340 have positive Δδ/ΔT values at both pH conditions. Interestingly, some residues (V295,A297, E318, andN352) at pH = 6.0 have significantly different Δδ/ΔT values compared with those at pH = 8.0 (Figure 1(b)). For example, the Δδ/ΔT value of N352 at pH = 6.0 is 0.8 ppb/K, but this value became −4.7 ppb/K at pH = 8.0 (Figure 1(b)). The longitudinal R1 relaxation rates and transverse R2 relaxation rates relate to the rotational correlation times that depend on the molecular radius, solvent viscosity, and temperature. The R1 decreases with molecular size for large molecules (over 1 kDa) while R2 increases. 8 Significantly reducedR2 and heteronuclear Overhauser effect (NOE) values indicate the fast timescale (ps–ns) motion of proteins such as backbone fluctuation and sidechain rotation. The R1 and R2 rates for uniformly N-labeled hAFL were measured at pH = 6.0 and compared with those determined at pH = 8.0, which were reported previously, to gain insight into the pHdependent dynamic motions of hAFL. R1 values of hAFL are fairly uniform throughout the proteins and increase with temperature at both pHs (Figure 2(a) and Table 1). At pH = 6.0, residues K334 and E341 showed significantly elevated R1 values at 5 C compared to other residues except both termini (Figure 2(a)). The R2 values showed larger deviation from the average values as the temperature decreased (Figure 2(b) and Table 1). Interestingly, the average R2 of hAFL at pH = 6.0 is larger than that at pH = 8.0 at 25 C, Note DOI: 10.1002/bkcs.10569 S.-R. Choi et al. BULLETIN OF THE KOREAN CHEMICAL SOCIETY