Structural Basis of Telomerase Inhibition by the Highly Specific BIBR1532

Abstract

SUMMARY BIBR1532 is a highly specific telomerase inhibitor, however the molecular basis for inhibition is unknown. Here we present the crystal structure of BIBR1532 bound to Tribolium castaneum catalytic subunit of telomerase (tcTERT). BIBR1532 binds to a conserved hydrophobic pocket (FVYL motif) on the outer surface of the thumb domain. The FVYL motif is near TRBD residues that bind the activation domain (CR4/5) of hTER. RNA binding assays show that the human TERT (hTERT) thumb domain binds the P6.1 stem loop of CR4/5 in vitro. hTERT mutations of the FVYL pocket alter wild type CR4/5 binding and cause telomere attrition in cells. Furthermore, the hTERT FVYL mutations V1025F, N1028H and V1090M are implicated in dyskeratosis congenita and aplastic anemia, further supporting the biological and clinical relevance of this novel motif. We propose that CR4/5 contacts with the telomerase thumb domain contribute to telomerase ribonucleoprotein (RNP) assembly and promote enzymatic activity.

Structural Analysis Reveals the Deleterious Effects of Telomerase Mutations in Bone Marrow Failure Syndromes

“…Contrary to the mutations that contribute to hybrid binding, N1028H and V1090M showed a slight (1.5-fold) decrease in hRNADNA binding affinity, which is within the margin error. It is worth noting that in the full-length TERT, the hRNADNA hybrid makes additional contacts with the TRBD and RT domains (2,6). We therefore expect a higher affinity for the hRNADNA in the context of the full-length protein, consistent with what has been previously reported (26).…”

“…Another highly conserved region of the hThumb is the FVYL pocket formed by the loops that connect motifs E-I, E-II, and E-III and was previously suggested to bind the P6.1 stem loop of CR4/5 (6). The FVYL pocket is located at the interface of the TRBD-thumb domain and is ϳ12 Å away from the nearest TRBD residue.…”

“…The protein subunit, TERT, 2 consists of several domains (TEN, TRBD, RT (fingers and palm), and thumb) organized into a closed, ring configuration, generating a large cavity in the interior of the ring and where the RNA template and the DNA bind during telomere elongation (2)(3)(4)(5). The closed configuration of the TERT ring is stabilized by extensive interactions between the thumb and TRBD domains as well as by protein-RNA interactions (3,6). Several invariable motifs in the interior cavity of the TERT ring coordinate the RNA template and telomeric overhang (RNADNA hybrid) and position the 3Ј end of the DNA for catalysis (2,3).…”

“…The telomerase activation domain consists of the stem loops P6a/b, P6.1, and P5 (15) and is coordinated by the VSR motif of TRBD and motifs E-II and E-III (FVYL pocket) of the thumb domain (6,9,10). Although CR4/5 is primarily coordinated by the TRBD, its stem loop P6.1 extends across the TRBD-thumb interface and binds the FVYL pocket of the thumb domain (6).…”

“…Although CR4/5 is primarily coordinated by the TRBD, its stem loop P6.1 extends across the TRBD-thumb interface and binds the FVYL pocket of the thumb domain (6). Extensive interactions between the CR4/5 and the terminal domains of the TERT ring most likely stabilize the telomerase ribonucleoprotein complex and lock the closed ring configuration of TERT during telomere elongation.…”