Dimerization and Threonine-Dependent Stabilization Govern Human YRDC Catalysis

Technical Summary: Dimerization and Threonine-Dependent Stabilization Govern Human YRDC Catalysis

Problem Statement
The enzyme YRDC catalyzes the first step in the biosynthesis of N⁶-threonylcarbamoyladenosine (t⁶A37), a critical post-transcriptional modification found at position 37 of specific mitochondrial and cytoplasmic tRNAs. This modification is essential for translation fidelity and efficiency. Mutations in the human YRDC gene are linked to Galloway-Mowat syndrome (GAMOS), a severe neuro-renal disorder with a poor prognosis. Despite the enzyme's clinical relevance and its role in cancer cell survival, the precise catalytic mechanism, the order of substrate binding, the structural basis for amino acid selectivity, and the molecular consequences of disease-associated mutations remained undefined prior to this study.

Methodology
The authors employed a multidisciplinary approach combining protein biophysics, structural biology, mass spectrometry, and enzymology to characterize a truncated human YRDC construct (ΔN43, lacking the N-terminal 43 residues).

• Biophysical Characterization: Differential scanning fluorimetry (DSF) and circular dichroism (CD) spectroscopy were used to assess thermal stability ($T_m$), secondary structure composition, and ligand-induced stabilization.
• Kinetic Analysis: An LC-MS assay quantifying AMP formation (a proxy for the unstable intermediate threonylcarbamoyl-AMP, TC-AMP) was established to determine Michaelis-Menten parameters ($K_m$, $k_{cat}$, $k_{cat}/K_m$) for ATP, L-threonine, and bicarbonate.
• Structural and Oligomeric State Analysis: Native mass spectrometry, mass photometry (MP), native PAGE, and crosslinking mass spectrometry (using BS3) were utilized to determine the oligomeric state of wild-type (WT) and mutant proteins.
• NMR Spectroscopy: Saturation transfer difference (STD) NMR and heteronuclear NMR were used to investigate direct binding interactions and to test for the existence of a non-enzymatic threonine-carbamate intermediate.
• Mutagenesis: Site-directed mutagenesis was performed on residues predicted to be in the active site (based on homology with Sulfolobus tokodaii SUA5) and on residues associated with GAMOS (A41V, I178T, ΔL222).

Key Results

1. L-Threonine as a Gatekeeper Ligand:

   • DSF and CD data reveal that L-threonine acts as a "gatekeeper," binding with millimolar affinity ($K_D \approx 31.1$ mM) and inducing significant thermal stabilization of the protein.
   • Threonine binding is a prerequisite for high-affinity binding of ATP (reducing $K_D$ from ~2 mM to ~77 µM) and bicarbonate. Without threonine, ATP and bicarbonate binding is negligible or undetectable.
   • While other amino acids (L-Cys, L-Ser, L-Ala, L-Val) can bind and stabilize YRDC to varying degrees, L-threonine provides the most effective stabilization and product formation.

2. Catalytic Mechanism and Substrate Specificity:

   • Kinetic analysis confirms that YRDC can utilize alternative amino acids to form carbamoyl-AMP adducts, but L-threonine is the preferred substrate with superior catalytic efficiency.
   • NMR data (specifically the absence of a threonine-carbamate adduct in the absence of the enzyme and the lack of ADP accumulation) support a mechanism where YRDC forms a quaternary complex with L-threonine, ATP, and bicarbonate directly on the enzyme surface. This refutes hypotheses suggesting a pre-formed threonine-carbamate intermediate in solution or a multi-step ATP activation prior to TC-AMP formation.

3. Dimerization is Essential for Function:

   • Native mass spectrometry, mass photometry, and crosslinking experiments demonstrate that WT human YRDC exists predominantly as a dimer (~50 kDa) in solution.
   • GAMOS-associated mutations (A41V, I178T, ΔL222) disrupt this quaternary structure. While WT forms a stable dimer, these mutants exhibit a shift toward monomers and aberrant higher-order oligomers (trimers, tetramers).
   • Crosslinking identified Lys118 and Lys73 as being in close proximity within the dimer interface.

4. Impact of Mutations on Stability and Activity:

   • Active Site Mutants: Mutations in residues predicted to contact substrates (e.g., T46A, R130A) largely abolished catalytic activity and often resulted in unstable, unfolded proteins that could not bind ligands effectively.
   • GAMOS Mutants: These mutations caused severe structural destabilization (increased disordered/coil content, reduced $\beta$-sheet content) and loss of dimerization.
     • YRDCA41V and YRDCΔL222 retained near-WT catalytic turnover ($k_{cat}$) but showed reduced ATP affinity, likely due to global destabilization affecting the active site geometry.
     • YRDCI178T exhibited a significant reduction in $k_{cat}$ (six-fold lower than WT) while maintaining near-WT ATP affinity, suggesting a specific defect in the catalytic step or the maintenance of the active dimeric conformation.
   • Crucially, GAMOS mutants failed to undergo ligand-induced stabilization, indicating they exist in a predominantly disordered state that cannot be rescued by substrate binding.

Significance and Claims
The authors conclude that human YRDC catalysis is governed by two critical control points: dimerization and threonine-dependent conformational stabilization. The study establishes that L-threonine binding is the initial step that orders the protein structure, enabling the subsequent binding of ATP and bicarbonate to form a productive quaternary complex.

The paper claims that GAMOS-linked mutations cause disease not merely by disrupting the active site, but by destabilizing the protein's fold and preventing the formation of the functional dimer. This loss of quaternary structure leads to a non-functional enzyme, explaining the severe phenotypic consequences of these mutations. The work provides a biophysical foundation for understanding the molecular pathology of GAMOS and suggests that the dimer interface and threonine-binding pocket are potential targets for designing selective YRDC inhibitors (for cancer therapy) or stabilizers (for treating GAMOS).