Structure of the essential diversity-generating retroelement protein bAvd and its functionally important interaction with reverse transcriptase

Abstract

Diversity-generating retroelements (DGRs) are the only known source of massive protein sequence variation in prokaryotes. These elements transfer coding information from a template region (TR) through an RNA intermediate to a protein-encoding variable region. This retrohoming process is accompanied by unique adenine-specific mutagenesis and, in the prototypical BPP-1 DGR, requires a reverse transcriptase (bRT) and an accessory variability determinant (bAvd) protein. To understand the role of bAvd, we determined its 2.69 Å resolution structure, which revealed a highly positively charged pentameric barrel. In accordance with its charge, bAvd bound both DNA and RNA, albeit without a discernable sequence preference. We found that the coding sequence of bAvd functioned as part of TR but identified means to mutate bAvd without affecting TR. This mutational analysis revealed a strict correspondence between retrohoming and interaction of bAvd with bRT, suggesting that the bRT-bAvd complex is important for DGR retrohoming.

Figure 1. Mutagenic retrohoming

a. Information transfer from the TR to the VR via mutagenic retrohoming. Functional elements at the 3' ends of the VR and TR: blue circles, the GC-only sequence; red and green diamonds, the IMH and IMH* elements, respectively. The hairpin/cruciform structure following the VR is shown as well. b. Target-primed reverse transcription. Cleavage of the VR lower strand in the GC-only sequence would enable that strand to act as a primer for reverse transcription by bRT, with an RNA containing the TR acting as a template.

Figure 2. bAvd structure

a. bAvd pentamer in ribbon representation, with each protomer colored differently. b. Cross-section of the bAvd barrel in molecular surface representation, colored by the electrostatic potential, ranging from −10 kT/e (red) to +10 kT/e (blue). c. Narrow end of the bAvd barrel in ribbon representation. See also Fig. S1. d. Narrow end of the bAvd barrel colored according to electrostatic potential, in molecular surface representation, as in panel b. See also Fig. S3.

Figure 3. Four helix bundle and pentameric interface

a. A bAvd protomer in cartoon representation (N- to C-terminus, blue to red in rainbow coloring). Helices α1 and α2 are at a 20° angle to helices α3 and α4. b. Hydrophobic residues buried in the interprotomer interface. See also Fig. S2. c. Hydrogen bonding between protomers.

Figure 4. Patterns of conservation

Residues conserved within the DGR Avd family mapped onto the surface of bAvd (color scale below). Only one of the protomers is colored, the others are in yellow. Top, the side of the barrel. Middle, narrow end of the barrel. Bottom, wide end of the barrel.

Figure 5. Functionally significant bAvd residues

a. Kanamycin-resistance retrohoming assay. VR containing aph3' Ia deleted of its 6 C-terminal residues (kan) is on a phage lysogen, and the TR containing the 3' end of aph3' Ia (kan3') on the pMX-Km1 plasmid. Functional elements are as in Figure 1. Derivatives of pMX-Km1 containing only 3' portions of avd are indicated below the schematic of the plasmid. The pFHA-avd plasmid, which provides intact avd in trans, is on the top right. P_FHA is the Bordetella fhaB promoter, and Ori is the origin of replication sequence. b. Retrohoming frequency resulting from cotransformation of pFHA-avd with pMX plasmids expressing intact avd (Km1), catalytically defective bRT (Km1RT^—), or avd containing only 48 (Δavd), 53 (T+53), 76 (T+76), 115 (T+115), 200 (T+200), or 300 (T+300) bp of 3' end of avd. Results are the average of two independent experiments or four in the case of Km1 and Km1RT^—; error bars represent standard deviations. c. Top, homing frequency resulting from cotransformation of pMX-T+300 and pFHA-avd expressing wild-type bAvd (WT), no bAvd (-bAvd), bAvd R19A, P35A, R36A, K37A, E43A, Q64A, R79A, or R83A. Results are the average of three independent experiments and error bars represent standard deviations. Bottom, western blot of bAvd using antibodies that recognize a His-tag incorporated at the C-terminus of bAvd. R19A has a minor band of smaller size, which could be a degradation product or caused by internal translational initiation. d. Circular dichroism spectra at 4 °C (black) and 37 °C (gray) of wild type bAvd (♦), P35A (▲), R36A (●), K37A (■), R79A (−), and R83A (+). Mean residue ellipticity (MRE) values were measured from 195 nm to 250 nm.

Figure 6. Interactions of bAvd

a. Upper panel, Ethidium bromide-stained EMSA of bAvd at varying concentrations (indicated above lanes as µM of pentamer) incubated with 0.5 µM of TR RNA (ssRNA), VR DNA (ssDNA), a heteroduplex of VR DNA and TR RNA (DNA:RNA), a homoduplex of VR DNA (dsDNA), and a homoduplex of a VR DNA variant engineered to favor the formation of a hairpin/cruciform structure (cruci-DNA). Lower panel, the same experiment was carried out with random RNA and DNA sequences; in the case of the cruci-DNA, a non-functional loop sequence was used. Arrows indicate the positions of shifted bands. See also Fig. S6. b. Association of wild-type and mutant bAvd with His-bRT D138Q, as detected by a Ni²⁺-NTA coprecipitation assay and visualized by Coomassie-stained SDS-PAGE. Bound fractions shown here, unbound fractions in Figure S4d.