Hot-spot analysis to dissect the functional protein-protein interface of a tRNA-modifying enzyme

Abstract

Interference with protein-protein interactions of interfaces larger than 1500 Ų by small drug-like molecules is notoriously difficult, particularly if targeting homodimers. The tRNA modifying enzyme Tgt is only functionally active as a homodimer. Thus, blocking Tgt dimerization is a promising strategy for drug therapy as this protein is key to the development of Shigellosis. Our goal was to identify hot-spot residues which, upon mutation, result in a predominantly monomeric state of Tgt. The detailed understanding of the spatial location and stability contribution of the individual interaction hot-spot residues and the plasticity of motifs involved in the interface formation is a crucial prerequisite for the rational identification of drug-like inhibitors addressing the respective dimerization interface. Using computational analyses, we identified hot-spot residues that contribute particularly to dimer stability: a cluster of hydrophobic and aromatic residues as well as several salt bridges. This in silico prediction led to the identification of a promising double mutant, which was validated experimentally. Native nano-ESI mass spectrometry showed that the dimerization of the suggested mutant is largely prevented resulting in a predominantly monomeric state. Crystal structure analysis and enzyme kinetics of the mutant variant further support the evidence for enhanced monomerization and provide first insights into the structural consequences of the dimer destabilization.

Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB

“…For RNase III, only one of the two equivalent residues of the homodimer is mutated to Ala in the computational Ala scanning (that is, a mutant/wt heterodimer is used in the calculation of ΔΔGbind), while the experimental Ala mutagenesis analysis (see next section) necessarily involves a homodimer with a mutation in each subunit. To address this limitation of the calculation, we have estimated the change in binding free energy for a given mutant/mutant homodimer as the sum of the ΔΔGbind of the two alternative mutant/wt heterodimers [see Eq. (2), SI‐1].…”

“…To address this limitation of the computational methodology, and to be able to compare the results with experimental data, we estimated the change in binding free energy for a given mutant‐mutant RNase III homodimer as the sum of the ΔΔ G bind of the two alternative mutant/wt heterodimer forms: where X and X ′ are the two symmetry‐related RNase III residues, XA and X ′ A denote the corresponding single alanine mutants, and XA/X ′ A the double mutant. This additivity approximation assumes that the two single alanine mutations ( XA and X ′ A ) are functionally independent, in that the coupling or interaction free energy between the two residues ( X and X ′) is zero. Therefore, the ΔΔ G bind values provided in Table II and in Supporting Information Figure S12 (SI‐1) are an estimate of the actual values for the mutant/mutant RNase III homodimer.…”

Cross-Talk between Dnmt2-Dependent tRNA Methylation and Queuosine Modification

“…The eTGTs consist of two subunits that both are evolutionarily related to bTGT: the catalytic subunit queuine-tRNA-ribosyltransferase (QTRT1) and the accessory subunit queuine-tRNA ribosyltransferase domain containing 1 (QTRTD1, re-annotated as QTRT2) [28]. It is interesting to note that eTGT acts as a heterodimer, whereas bTGT functions as a homodimer [29]. In addition, although QTRT1 and QTRTD1 are related to bTGT, QTRTD1 apparently has lost its enzymatic activity, but still is essential for eTGT activity.…”

Austausch der Proteinkontaktflächen in der homodimeren tRNA‐Guanin‐Transglycosylase: ein Weg der funktionellen Regulation

“…[7] Überraschenderweise sind die hydrophoben Aminosäuren Va l282, Leu68 und Va l45, die das untere Ende der Ribose-34-Tasche flankieren, äußerst empfindlich gegen Wechselwirkungen mit den funktionellen Gruppen unserer Liganden, die sich bei Bindung an das Protein in Nachbarschaft zu diesen Resten befinden. [8,9] Wiruntersuchten mit nicht-denaturierender nano-ESI-Massenspektrometried as Monomer-zu-Dimer-Verhältnis in Lçsung und fokussierten die Studie besonders auf Va rianten des Proteins,die einzelne Punktmutationen von Aminosäuren aufweisen. Dieses Motiv verhindert den Zugang von Wasser zu einem essentiellen hydrophoben Cluster aus vier aromatischen Aminosäuren (Trp326, Ty r330, His333 und Phe92'), die von einem charakteristischen O-Ring-Motiv umgeben sind.…”

“…[9] Es wurde gezeigt, dass die Integritätd er räumlichen Anordnung der vier aromatischen Aminosäuren fürd ie Stabilitätd er Homodimerkontaktfläche essentiell ist. [8,9] Die substituierten lin-Benzoguanine 1-3 wurden nach einer Prozedur hergestellt, die bereits von uns publiziert wurde. [11,12] Schema 1z eigt die Synthese von 4-6.D as 2-(Nitro)amino-6-nitrobenzimidazol 7 wurde durch Nitrierung des 2-Aminobenzimidazols 8 (siehe Abschnitt S6.4 der SI für Details) erhalten.…”