Caught in motion: human NTHL1 undergoes interdomain rearrangement necessary for catalysis

Abstract

Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and β-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1.

OpenFold: Retraining AlphaFold2 yields new insights into its learning mechanisms and capacity for generalization

“… 2 PDB accession codes 7DQ9_A (Wei et al 2021), 7RDT_A (Carroll et al 2021), and 7LBU_A (Yu et al 2021)…”

Human endonuclease III/NTH1: focusing on the [4Fe–4S] cluster and the N-terminal domain

“…[2][3][4][5] Recently the three-dimensional structure of hNTH1 lacking the first 63 amino acids (hNTH1D63) was determined. 6 In this structure the first twenty amino acids are not visible in the electron density map, indicating a distinct flexibility of the NTD. Overall, the domain composition and organization of hNTH1D63 are similar to that of bacterial EndoIIIs, [7][8][9] however, domains A and B adopt an 'open' configuration in hNTH1D63, which is distinct from the 'closed' bacterial EndoIIIs (Fig.…”

“…These fits clearly indicate that hNTH1 adopts 3A) as reported recently in the crystal structure of hNTH1D63. 6 Carroll et al proposed that this alternative conformation may relate to the longer linker region connecting domains A and B in hNTH1 compared to its bacterial homologues. 6 However, this model is not supported by our finding that the full-length enzyme, hNTH1, which also bears a longer linker, adopts a 'closed' conformation (Fig.…”

“…The unique conformation of hNTH1Δ63 was attributed to a more flexible linker connecting domains A and B, which when substituted by the shorter linker found in E. coli EndoIII (EcEndoIII) resulted in a ‘closed’ conformation. 6 The transition from an ‘open’ to a ‘closed’ conformation was proposed to be induced upon DNA binding (between domain A and B), as observed in a DNA-bound bacterial EndoIII. 8 The role of the [4Fe–4S] cluster in EndoIII enzymes is still under debate.…”

Disentangling Unusual Catalytic Properties and the Role of the [4Fe-4S] Cluster of Three Endonuclease III from the Extremophile D. radiodurans

“…Domain A consists of 4 alphahelices, the N and C termini, a [4Fe-4S] cluster loop (FCL) and a [4Fe-4S] cluster; the domain B consist of six alpha-helices and two DNA intercalating loops (DIL). Recently the crystal structure of the catalytic domain of human EndoIII (hNTH1) was also determined, revealing the same organization (A and B) as its bacterial homologues [6]. Thus, results from studies of bacterial EndoIII enzymes may also be relevant for understanding the molecular mechanisms underlying the function of the human enzyme.…”