NMR solution structures of Runella slithyformis RNA 2′-phosphotransferase Tpt1 provide insights into NAD+ binding and specificity

Abstract

Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2′-PO4 to NAD+ yielding RNA 2′-OH and ADP-ribose-1′,2′-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD+. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD+-bound states. ITC measurements of RslTpt1 binding to NAD+ (KD ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD+-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP+, which contains an adenylate 2′-PO4 (mimicking the substrate RNA 2′-PO4), binds with lower affinity (KD ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD+ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD+ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction.

Structural basis for Tpt1-catalyzed 2′-PO4transfer from RNA and NADP(H) to NAD+

“…4 C ; magenta dashed line) and reinforces the exclusion of NAAD as a substrate. Comparison of the conformation of NAD + in the PhoTpt1 crystal structure to that calculated by solution NMR for NAD + bound to Runella Tpt1 ( 20 ) reveals that whereas the AMP moieties are virtually identical in conformation and positions within the NAD + lobe, the nicotinamide riboside moieties adopt opposite orientations, whereby the nicotinamide in the calculated NMR structure overlies the ribose in the crystal structure and the ribose in the NMR structure overlies the nicotinamide in the crystal structure. Although it is conceivable that NAD + can explore multiple states on the enzyme before assuming a catalytically competent one, we regard the crystal structure, with its unambiguous electron density for NAD + , as the on-pathway state of the Tpt1·NAD + substrate complex.…”

“…For example, Tpt1 enzymes catalyze transfer of ADP-ribose from NAD + to 5′-monophosphate termini of DNA or RNA to form a 5′-phospho-ADP-ribosyl cap structure ( 15 , 23 ). We were attracted to the idea that Tpt1 might act on a phospho-metabolite, in light of biophysical evidence (via NMR and isothermal titration calorimetry) that Runella Tpt1 can bind NADP + in its RNA lobe ( 20 ). NADP + is a plausible substrate for Tpt1 insofar as it possesses a 5′-phospho-adenosine-2′-PO 4 moiety.…”

“…We surmise that the RNA lobe with its bound 2′-phosphodonor substrate must undergo a slight movement toward the NAD + lobe in the context of a bona fide Michaelis complex, presumably by flexion around the protease-sensitive interdomain linker segment, to close the distance between the 2′-PO 4 and C1″. Solution NMR studies of Runella Tpt1 had established that the RNA and NAD + lobes display significant interlobe mobility ( 20 ).…”

“…Tpt1 proteins consist of an N-terminal RNA lobe and a C-terminal NAD + lobe separated by a protease-sensitive hinge ( 13 , 19 – 21 ). Solution NMR studies of Runella Tpt1 revealed significant interlobe mobility in the absence of substrate, and in the presence of NAD + ( 20 ).…”

“…Tpt1 proteins consist of an N-terminal RNA lobe and a C-terminal NAD + lobe separated by a protease-sensitive hinge ( 13 , 19 – 21 ). Solution NMR studies of Runella Tpt1 revealed significant interlobe mobility in the absence of substrate, and in the presence of NAD + ( 20 ). Insights into the mechanism of the transesterification step emerged from a crystal structure of Clostridium thermocellum (Cth) Tpt1 in a product-mimetic complex that contained ADP-ribose-1″-PO 4 in the NAD + lobe, notwithstanding that no exogenous ligand was added during crystallization ( 21 ).…”