Conformational dynamics of the Escherichia coli DNA polymerase manager proteins UmuD and UmuD'

Abstract

The expression of Escherichia coli umuD gene products is upregulated as part of the SOS response to DNA damage. UmuD is initially produced as a 139-amino-acid protein, which subsequently cleaves off its N-terminal 24 amino acids in a reaction dependent on RecA/single-stranded DNA, giving UmuD'. The two forms of the umuD gene products play different roles in the cell. UmuD is implicated in a primitive DNA damage checkpoint and prevents DNA polymerase IV-dependent -1 frameshift mutagenesis, while the cleaved form facilitates UmuC-dependent mutagenesis via formation of DNA polymerase V (UmuD'(2)C). Thus, the cleavage of UmuD is a crucial switch that regulates replication and mutagenesis via numerous protein-protein interactions. A UmuD variant, UmuD3A, which is noncleavable but is a partial biological mimic of the cleaved form UmuD', has been identified. We used hydrogen-deuterium exchange mass spectrometry (HXMS) to probe the conformations of UmuD, UmuD', and UmuD3A. In HXMS experiments, backbone amide hydrogens that are solvent accessible or not involved in hydrogen bonding become labeled with deuterium over time. Our HXMS results reveal that the N-terminal arm of UmuD, which is truncated in the cleaved form UmuD', is dynamic. Residues that are likely to contact the N-terminal arm show more deuterium exchange in UmuD' and UmuD3A than in UmuD. These observations suggest that noncleavable UmuD3A mimics the cleaved form UmuD' because, in both cases, the arms are relatively unbound from the globular domain. Gas-phase hydrogen exchange experiments, which specifically probe the exchange of side-chain hydrogens and are carried out on shorter timescales than solution experiments, show that UmuD' incorporates more deuterium than either UmuD or UmuD3A. This work indicates that these three forms of the UmuD gene products are highly flexible, which is of critical importance for their many protein interactions.

Fig. 1

Cleavage of UmuD removes the N-terminal 24 amino acids and dictates different cellular functions. (a) A homology model of the UmuD₂ dimer which functions in a primitive DNA damage checkpoint and (b) NMR structure of UmuD′₂ dimer (PBD code: 1I4V31) which facilitates UmuC-dependent TLS. One monomer is in blue; the other is in red in both images. The first 24 residues at the N-termini are shown in yellow. The residues mutated to alanines in UmuD3A [Thr14 (purple), Leu17 (cyan), and Phe18 (green)] are shown in space-filling rendering.

Fig. 2

Melting curves of UmuD (blue), UmuD′ (green), and UmuD3A (pink) show the distinctive unfolding transitions of each protein. The T_ms of UmuD and UmuD′ are each 60 °C. The T_m of UmuD3A is 59 °C. Additionally, the UmuD melting curve shows a second melting transition at a T_m of approximately 30 °C.

Fig. 3

Relative deuterium uptake as a function of time for intact UmuD (blue), UmuD3A (pink) and UmuD′ (green). Intact protein exchange analyzed at different deuterium exchange time points between 0 and 480 min. The data shown here are the average of two experiments. The total number of exchangeable backbone amide hydrogens in intact UmuD and UmuD3A is 128 and in UmuD′ is 108.

Fig. 4

Deuterium incorporation of UmuD and UmuD′ as a function of time. (a) Deuterium incorporation information for UmuD is mapped on to its homology model. (b) Deuterium incorporation data for UmuD′ are mapped on the “filament” dimer of UmuD′ crystal structure. For both UmuD and UmuD′, the protein is shown in two orientations, with the top orientation turned 90 degrees toward the observer on the y-axis. The relative percentage deuterium incorporation for all monitored residues of UmuD (see also Supplementary Fig. 4) is shown at the times indicated. The color code is shown at the bottom of the figure. The UmuD data in this figure represents each frame of Supplementary Movie S1. The UmuD′ data in this figure represents each frame of Supplementary Movie S2. Regions colored gray represent residues where deuterium levels were not determined.

Fig. 5

Comparison of deuterium exchange in (a) UmuD and UmuD′ and in (b) UmuD and UmuD3A. The deuterium uptake curves for representative peptides (the complete dataset is in Supplementary Fig. 4) are shown at the top (UmuD in blue, UmuD3A in pink, and UmuD′ in green). The location of each peptide, according to the labels a1-3 and b1-4, is shown on the homology model of UmuD at the bottom, in two orientations: from the “front” and from the “top”. Obvious changes in red were defined as a difference between deuterium exchange of 1.0 Da or greater, observed in at least one time point. Subtle changes in yellow were 0.4–1.0 Da. No changes, shown in gray, were defined as differences of 0.0–0.4 Da. The first N-terminal 24 residues are shown in (a) in blue. In (b), the residues mutated to alanines in UmuD3A [Thr14 (purple), Leu17 (cyan) and Phe18 (green)] are shown as spheres.

Fig. 6

Gas-phase HX of UmuD proteins. (a) Mass spectra recorded of UmuD (spectra i and ii) and UmuD′ (spectra iii and iv) in the absence and presence of labeling ND₃ gas (4.2 × 10⁻³ mbar). (b) Bar chart of the deuterium uptake during 0.3 ms of charge states 6+, 7+, and 8+ of UmuD′ (green) UmuD3A (pink) and UmuD (blue) in the presence of ND₃ gas (4.2 × 10⁻³ mbar). The deuterium uptake of a gas-phase HX control peptide (Leucine enkephalin), which was co-infused with the UmuD proteins, is shown in white bars. The error bars represent the standard deviation of triplicate measurements for UmuD′. Data for UmuD and UmuD3A were acquired in duplicate. (c) Probing sub-millisecond conformational dynamics of UmuD proteins by time-resolved gas-phase deuterium uptake of the [M+7H]⁷⁺ charge state of UmuD′ (green triangles), UmuD3A (pink squares) and UmuD (blue diamonds) upon exchange with ND₃ gas (1.6 × 10⁻³ mbar). Note, gas-phase HX at such short timescales primarily occurs at labile and exposed hydrogens in the side-chain positions of the protein.