Dynamics of the HD regulatory subdomain of PARP-1; substrate access and allostery in PARP activation and inhibition

Abstract

PARP-1 is a key early responder to DNA damage in eukaryotic cells. An allosteric mechanism links initial sensing of DNA single-strand breaks by PARP-1's F1 and F2 domains via a process of further domain assembly to activation of the catalytic domain (CAT); synthesis and attachment of poly(ADP-ribose) (PAR) chains to protein sidechains then signals for assembly of DNA repair components. A key component in transmission of the allosteric signal is the HD subdomain of CAT, which alone bridges between the assembled DNA-binding domains and the active site in the ART subdomain of CAT. Here we present a study of isolated CAT domain from human PARP-1, using NMR-based dynamics experiments to analyse WT apo-protein as well as a set of inhibitor complexes (with veliparib, olaparib, talazoparib and EB-47) and point mutants (L713F, L765A and L765F), together with new crystal structures of the free CAT domain and inhibitor complexes. Variations in both dynamics and structures amongst these species point to a model for full-length PARP-1 activation where first DNA binding and then substrate interaction successively destabilise the folded structure of the HD subdomain to the point where its steric blockade of the active site is released and PAR synthesis can proceed.

PARP Power: A Structural Perspective on PARP1, PARP2, and PARP3 in DNA Damage Repair and Nucleosome Remodelling

“…[53], 4TVJ [115]) contains a unique insertion compared to PARP1 (red, PDB 7AAD, bound to olaparib [109]) or PARP3 (blue, PDB 4GV2, bound to ME0354 [116]). This is suggested to increase branching of PAR chains by PARP2 [112]; (E) Close-up of the NAD + binding pocket of PARP1 (red, olaparib-bound, PDB 7AAD [109]). The catalytic triad involving H862, Y896, and E988 and the residues interacting with PARPi, G863-S864, and S904, are indicated.…”

“…Only through correct placement of Zn1 and Zn2 domains, on a SSB in the observed directionality, do Zn3 and WGR form the "landing pad" assembly (Figure 4, inset ii) on which HD can dock and subsequently become partially destabilised to activate ), PARP2 (white, olaparib-bound, PDB 4TVJ [115]), and PARP3 (blue, ME0354-bound, PDB 4GV2 [116]) with highlighted features. (A) A folded HD (white) occludes the NAD + binding pocket in the PARP1 ART domain (red, PDB 7AAB, PARP1 with NAD + analogue EB-47 bound [109]); (B) Subtle movement of parts of the HD domains in response to DNA binding after superposition of the ART domain only. Left: HD of apo PARP1 before (red, PDB 7AAA [109]) and after (yellow, PDB 4DQY [34]) DSB model binding.…”

“…However, during self-assembly the decrease in entropy allows for enough free state energy to partly destabilise αB and part of αF of HD of the CAT domain, thereby allowing access for NAD + [ 57 , 108 ]. A crystallographic snapshot of this subtle movement is depicted in the superposition of crystal structures of ART domains of DNA-free and DNA-bound PARP enzymes, visualising the introduction of a kink in αF [ 109 ] at D766 (PARP1)/E335 (PARP2) ( Figure 3 B). Section 3.1 goes into this process in more detail including the dynamics measured in helices within the HD.…”

“…These PARP1 mutants with either the HD completely removed (ΔHD [ 57 ]) or a L713F mutation [ 118 ] feature increased DNA-independent activity that mimics DNA binding and allosteric activation. Using HDX-MS, the αB helix and part of the αF helix portion of the HD were shown to experience 1000-fold faster exchange [ 57 , 109 , 118 ]. Additionally, using NMR dynamics experiments significant amide proton exchange was detected in αB, αD, and αF of the HD, while Nuclear Overhauser Effect Spectroscopy (NOESY) experiments showed that the helices were still substantially intact (possibly with the exception of the HD in PARP1 when complexed with the largest inhibitor EB-47) [ 109 ].…”

“…ME0354-bound, PDB 4GV2[116]) with highlighted features. (A) A folded HD (white) occludes the NAD + binding pocket in the PARP1 ART domain (red, PDB 7AAB, PARP1 with NAD + analogue EB-47 bound[109]); (B) Subtle movement of parts of the HD domains in response to DNA binding after superposition of the ART domain only. Left: HD of apo PARP1 before (red, PDB 7AAA[109]) and after (yellow, PDB 4DQY[34]) DSB model binding.…”