The Importance of Charge in Perturbing the Aromatic Glue Stabilizing the Protein–Protein Interface of Homodimeric tRNA-Guanine Transglycosylase

Abstract

Bacterial tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the modified tRNA nucleoside queuosine present in the anticodon wobble position of tRNAs specific for aspartate, asparagine, histidine and tyrosine. Inactivation of the tgt gene leads to decreased pathogenicity of Shigella bacteria. Therefore, Tgt constitutes a putative target for Shigellosis drug therapy. Since only active as homodimer, interference with dimer-interface formation may, in addition to active-site inhibition, provide further means to disable this protein. A cluster of four aromatic residues seems important to stabilize the homodimer. We mutated residues of this aromatic cluster and analyzed each exchange with respect to dimer and thermal stability or enzyme activity applying native mass spectrometry, thermal shift assay, enzyme kinetics, and X-ray crystallography. Our structural studies indicate strong influence of pH on homodimer stability. Obviously, protonation of a histidine within the aromatic cluster promotes the collapse of an essential structural motif within the dimer interface at slightly acidic pH.

Targeting a Cryptic Pocket in a Protein–Protein Contact by Disulfide-Induced Rupture of a Homodimeric Interface

“…One monomer unit of the enzyme performs the catalytic base exchange reaction while the other one is required to place the bound tRNA substrate in the correct orientation. This mechanism has been analyzed in detail by enzyme kinetics, mutational studies, native mass spectrometry, and crystal structure analyses. − …”

“…To better understand the driving forces responsible for the assembly and stability of the homodimer interface, we performed mutational studies of the interface-forming residues − and launched spiking or twist-inducing active-site ligands into the interface region to perturb the formation of the homodimer. Native nanoelectrospray ionization mass spectrometry (nanoESI-MS) indicated the actual ratio of the monomer–dimer equilibrium in solution and additional crystal structure analyses elucidated the geometrical changes resulting from the induced perturbances.…”

“…The stability of the symmetrical homodimer interface, which spans more than 1600 Å 2 , is stabilized by a patch of four aromatic amino acids, Trp326, Tyr330, His333, and Phe92′, contributed by the two dimer mates (residue of second subunit indicated by ′, Figure a). The mutation of any of the four aromatic residues by a nonaromatic amino acid results in drastic loss of homodimer stability. − Whereas the wild type is nearly exclusively present in the dimeric state, for some of the mutated variants, only minor to hardly any dimer formation is observed in solution. The latter evidence is based on nanoESI-MS studies, which show a concentration-dependent increase of dissociation to monomeric state.…”

“…We hypothesized that this loop is crucial for the establishment of the interface. Supposedly, the loop is disordered in solution and adopts an ordered geometry in the unperturbed wild-type interface in order to operate as an additional shield to prevent water access, which otherwise would interfere with the aromatic hot spot. ,, In destabilized mutated variants and complexes with spiking and twist-inducing ligands, the loop–helix motif adopts a deviating conformation in the interface region. This motivated us to envision a strategy to raise small-molecule binders against this motif to alter the loop geometry into a state incompatible with interface formation.…”

“…Thus, we only modulated a strong and stable wild-type protein–protein contact into a weak crystal packing contact in the variants. This observation is an example for why it is so difficult to develop reliable descriptors to discriminate permanent PPIs from transient crystal packing contacts by analyzing crystallographic data only. , Furthermore, with increasing protein concentration, dimer formation is favored under equilibrium conditions as indicated by nanoESI-MS. , Upon crystallization, the local protein concentration next to the formed crystal nucleus increases significantly, thus, favoring dimer association even for variants with rather weak dimer stability. Thus, the development of small-molecule modulators against a homodimer is much more delicate than against a heterodimer where the individual components forming the complex can be separately studied and experimentally manipulated …”