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The weak interdomain coupling observed in the 70 kDa subunit of human replication protein A is unaffected by ssDNA binding.

ABSTRACT: Replication protein A (RPA) is a heterotrimeric, multi-functional protein that binds single-stranded DNA (ssDNA) and is essential for eukaryotic DNA metabolism. Using heteronuclear NMR methods we have investigated the domain interactions and ssDNA binding of a fragment from the 70 kDa subunit of human RPA (hRPA70). This fragment contains an N-terminal domain (NTD), which is important for hRPA70-protein interactions, connected to a ssDNA-binding domain (SSB1) by a flexible linker (hRPA70(1-326)). Correlation analysis of the amide (1)H and (15)N chemical shifts was used to compare the structure of the NTD and SSB1 in hRPA70(1-326) with two smaller fragments that corresponded to the individual domains. High correlation coefficients verified that the NTD and SSB1 maintained their structures in hRPA70(1-326), indicating weak interdomain coupling. Weak interdomain coupling was also suggested by a comparison of the transverse relaxation rates for hRPA70(1-326) and one of the smaller hRPA70 fragments containing the NTD and the flexible linker (hRPA70(1-168)). We also examined the structure of hRPA70(1-326) after addition of three different ssDNA substrates. Each of these substrates induced specific amide (1)H and/or (15)N chemical shift changes in both the NTD and SSB1. The NTD and SSB1 have similar topologies, leading to the possibility that ssDNA binding induced the chemical shift changes observed for the NTD. To test this hypothesis we monitored the amide (1)H and (15)N chemical shift changes of hRPA70(1-168) after addition of ssDNA. The same amide (1)H and (15)N chemical shift changes were observed for the NTD in hRPA70(1-168) and hRPA70(1-326). The NTD residues with the largest amide (1)H and/or (15)N chemical shift changes were localized to a basic cleft that is important for hRPA70-protein interactions. Based on this relationship, and other available data, we propose a model where binding between the NTD and ssDNA interferes with hRPA70-protein interactions.

SUBMITTER: Daughdrill GW

PROVIDER: S-EPMC55822 | biostudies-literature | 2001 Aug

REPOSITORIES: biostudies-literature

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The weak interdomain coupling observed in the 70 kDa subunit of human replication protein A is unaffected by ssDNA binding.

Daughdrill G W GW Ackerman J J Isern N G NG Botuyan M V MV Arrowsmith C C Wold M S MS Lowry D F DF

Nucleic acids research 20010801 15

Replication protein A (RPA) is a heterotrimeric, multi-functional protein that binds single-stranded DNA (ssDNA) and is essential for eukaryotic DNA metabolism. Using heteronuclear NMR methods we have investigated the domain interactions and ssDNA binding of a fragment from the 70 kDa subunit of human RPA (hRPA70). This fragment contains an N-terminal domain (NTD), which is important for hRPA70-protein interactions, connected to a ssDNA-binding domain (SSB1) by a flexible linker (hRPA70(1-326)). ...[more]

PMID: 11470885

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Project description:Replication protein A (RPA) is a heterotrimeric single-stranded DNA- (ssDNA) binding protein that can form a complex with the xeroderma pigmentosum group A protein (XPA). This complex can preferentially recognize UV-damaged DNA over undamaged DNA and has been implicated in the stabilization of open complex formation during nucleotide excision repair. In this report, nuclear magnetic resonance (NMR) spectroscopy was used to investigate the interaction between a fragment of the 70 kDa subunit of human RPA, residues 1-326 (hRPA70(1-326)), and a fragment of the human XPA protein, residues 98-219 (XPA-MBD). Intensity changes were observed for amide resonances in the (1)H-(15)N correlation spectrum of uniformly (15)N-labeled hRPA70(1-326) after the addition of unlabeled XPA-MBD. The intensity changes observed were restricted to an ssDNA-binding domain that is between residues 183 and 296 of the hRPA70(1-326) fragment. The hRPA70(1-326) residues with the largest resonance intensity reductions were mapped onto the structure of the ssDNA-binding domain to identify the binding surface with XPA-MBD. The XPA-MBD-binding surface showed significant overlap with an ssDNA-binding surface that was previously identified using NMR spectroscopy and X-ray crystallography. Overlapping XPA-MBD- and ssDNA-binding sites on hRPA70(1-326) suggests that a competitive binding mechanism mediates the formation of the RPA-XPA complex. To determine whether a ternary complex could form between hRPA70(1-326), XPA-MBD and ssDNA, a (1)H-(15)N correlation spectrum was acquired for uniformly (15)N-labeled hRPA70(1-326) after the simultaneous addition of unlabeled XPA-MBD and ssDNA. In this experiment, the same chemical shift perturbations were observed for hRPA70(1-326) in the presence of XPA-MBD and ssDNA as was previously observed in the presence of ssDNA alone. The ability of ssDNA to compete with XPA-MBD for an overlapping binding site on hRPA70(1-326) suggests that any complex formation between RPA and XPA that involves the interaction between XPA-MBD and hRPA70(1-326) may be modulated by ssDNA.

Project description:The relative orientation of the two variable domains, VH and VL , influences the shape of the antigen binding site, that is, the paratope, and is essential to understand antigen specificity. ABangle characterizes the VH -VL orientation by using five angles and a distance and compares it to other known structures. Molecular dynamics simulations of antibody variable domains (Fvs) reveal fluctuations in the relative domain orientations. The observed dynamics between these domains are confirmed by NMR experiments on a single-chain variable fragment antibody (scFv) in complex with IL-1? and an antigen-binding fragment (Fab). The variability of these relative domain orientations can be interpreted as a structural feature of antibodies, which increases the antibody repertoire significantly and can enlarge the number of possible binding partners substantially. The movements of the VH and VL domains are well sampled with molecular dynamics simulations and are in agreement with the NMR ensemble. Fast Fourier transformation of the ABangle metrics allows to assign timescales of 0.1-10?GHz to the fastest collective interdomain movements. The results clearly show the necessity of dynamics to understand and characterize the favorable orientations of the VH and VL domains implying a considerable binding interface flexibility and reveal in all antibody fragments (Fab, scFv, and Fv) very similar VH -VL interdomain variations comparable to the distributions observed for known X-ray structures of antibodies. SIGNIFICANCE STATEMENT: Antibodies have become key players as therapeutic agents. The binding ability of antibodies is determined by the antigen-binding fragment (Fab), in particular the variable fragment region (Fv). Antigen-binding is mediated by the complementarity-determining regions consisting of six loops, each three of the heavy and light chain variable domain VH and VL . The relative orientation of the VH and VL domains influences the shape of the antigen-binding site and is a major objective in antibody design. In agreement with NMR experiments and molecular dynamics simulations, we show a considerable binding site flexibility in the low nanosecond timescale. Thus we suggest that this flexibility and its implications for binding and specificity should be considered when designing and optimizing therapeutic antibodies.

Dataset Information

The weak interdomain coupling observed in the 70 kDa subunit of human replication protein A is unaffected by ssDNA binding.

Publications

The weak interdomain coupling observed in the 70 kDa subunit of human replication protein A is unaffected by ssDNA binding.

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