NMR Analysis of the Architecture and Functional Remodeling of a Modular Multidomain Protein, RPA

Abstract

Modular proteins with multiple domains tethered by flexible linkers have variable global archiectures. Using the eukaryotic ssDNA binding protein, Replication Protein A (RPA), we demonstrate that NMR spectroscopy is a powerful tool to characterize the remodeling of architecture in different functional states. The first direct evidence is obtained for the remodeling of RPA upon binding ssDNA, including an alteration in the availability of the RPA32N domain that may help explain its damage-dependent phosphorylation.The progression of DNA replication and repair requires the coordinated action of dynamic, multi-protein assemblies. We have previously proposed a critical role for proteins composed of multiple, flexibly attached domains in facilitating the action of these dynamic complexes 1 . Because these proteins can undergo intra-and inter-domain rearrangements, they are able to interact optimally with the ever-changing substrate landscape present during DNA processing. RPA is a prototypical modular multi-domain DNA processing protein with flexible linkers of various lengths (Figure 1). The trimer core is a compact assembly of three OB-fold domains (RPA70C/32D/14) to which is appended the disordered RPA32N functional domain, the RPA32C winged-helix domain, and the tandem RPA70AB and the RPA70N OB-fold NMR spectroscopy in solution is a powerful tool for characterizing proteins under conditions that preserve intrinsic dynamic properties. The advent of TROSY, CRINEPT and related experimental approaches 3 has vastly increased the upper limit of molecular masses accessible to study by NMR. Examples range from the globular malate synthase (82 kDa) to the oligomeric GroEL-GroES complex (872 kDa) to highly flexible domains from the ribosome (>2.5 MDa) 4 . In the case of RPA (116 kDa) and many other multi-domain proteins, modularity and interdomain flexibility are the critical properties that enable characterization of dynamic architectures by NMR.To illustrate the analytical framework, results are presented first for RPA70NAB (M r 45.8 kDa), which has an asymmetric arrangement with a 70-residue N-A linker and a 10-residue A-B linker (Figure 1). The 15 N-1 H TROSY-HSQC spectrum of 15 N-enriched RPA70NAB reveals the presence of over 370 of the 400 expected signals from 422 residues (Figure 2). The signals from each of the three domains appear in positions remarkably similar to those in NMR spectra of the three isolated domains ( Figure S1). Thus, all three domains are structurally independent and resonance assignments can be transferred from the isolated domains to RPA70NAB 5 . NMR is highly sensitive to differences in the degree of inter-domain flexibility; the signals from the A and B domains are substantially weaker than the signals from the N domain, even though all three domains are approximately the same mass ( Figure 2). The differences arise from the fact that although the A and B domains are structurally independent, the short A-B tether partially restricts their motions, whereas the much longer N-A tether e...

Domain structure of the DEMETER 5-methylcytosine DNA glycosylase

“…4). The lack of a requirement of the interdomain variable regions suggests that the three domains form a globular architecture and not a flexible "beads-on-a-string" arrangement, which has been shown to be important for other multidomain proteins involved in genome maintenance (33,34).…”

Solution NMR Structure of the C-terminal DNA Binding Domain of Mcm10 Reveals a Conserved MCM Motif

“…To gain insight into the extent of the interaction between the two domains, we took advantage of the sequence specific NMR assignments for both the ID and CTD. NMR is a powerful technique for studying protein structural dynamics, and has been applied recently to the highly modular 116-kDa RPA heterotrimer (43). The high protein concentrations required for NMR experiments prevented structural analysis of full-length XMcm10.…”

The Structure and Function of Replication Protein A in DNA Replication

“…RPA4 readily forms an alternative heterotrimeric complex with RPA1 and RPA3, called aRPA and is expressed in all human tissues, albeit at different levels. This alternate form of RPA failed to support replication in the in vitro ( a ) Schematic drawing of the three subunits (RPA1 = 70 kDa, RPA2 = 32 kDa, and RPA3 = 14 kDa), the folded domains ( thick colored rectangles ) and fl exible linkers de fi ned by limited proteolysis and NMR studies (Brosey et al 2009 ;Wold 1995, 1996 ) ( thin white rectangles ). The OB-folds are labeled A -F , individually colored, and this color code is used in all fi gures in this chapter.…”

“…Full-length heterotrimeric RPA was analyzed using NMR and gave rich insight into the folding and structural dynamics of this multidomain, fl exible protein (Brosey et al 2009 ) . The NMR spectra on the RPA trimer contained over 350 of the 550 expected signals domains F, A, B, wHLH and the N-terminus of RPA2.…”

“…TNRs can occur in non-coding sequences as well as within coding sequences. NMR structural studies of these repeat Bochkareva et al 2002 ;Brosey et al 2009 ;Deng et al 2007 ;Fanning et al 2006 ; sequences reveal and con fi rm the formation of hairpin and mismatched DNA duplex structures (Mariappan et al 1998 ) . In such instances when the replicative polymerase encounters a stable secondary structure that was not unwound by a helicase or DNA binding protein, it skips over the region resulting in a loss of genetic information, genome instability and disease progression.…”