Recognition of T-rich single-stranded DNA by the cold shock protein Bs-CspB in solution

Abstract

Cold shock proteins (CSP) belong to the family of single-stranded nucleic acid binding proteins with OB-fold. CSP are believed to function as 'RNA chaperones' and during anti-termination. We determined the solution structure of Bs-CspB bound to the single-stranded DNA (ssDNA) fragment heptathymidine (dT7) by NMR spectroscopy. Bs-CspB reveals an almost invariant conformation when bound to dT7 with only minor reorientations in loop beta1-beta2 and beta3-beta4 and of few aromatic side chains involved in base stacking. Binding studies of protein variants and mutated ssDNA demonstrated that Bs-CspB associates with ssDNA at almost diffusion controlled rates and low sequence specificity consistent with its biological function. A variation of the ssDNA affinity is accomplished solely by changes of the dissociation rate. 15N NMR relaxation and H/D exchange experiments revealed that binding of dT7 increases the stability of Bs-CspB and reduces the sub-nanosecond dynamics of the entire protein and especially of loop beta3-beta4.

Figure 1

The 2D ¹H/¹⁵N HSQC spectrum of free (black) and dT₇-bound (red) Bs-CspB in 50 mM Na-cacodylate, 3 mM MgCl₂, pH 7.0 at 15°C. The spectra were recorded at a Bs-CspB-concentration of 0.8 mM and a final dT₇ concentration of 1.2 mM.

Figure 2

Protection factors of amide protons from H/D exchange of free (hatched bars) and dT₇-bound (solid bars) Bs-CspB. Missing bars indicate amides, which got fully deuterated in the dead-time of the experiment (5 min). The solid bars on top represent the five β-strands of Bs-CspB.

Figure 3

Backbone superposition of the 18 lowest energy NMR structures of free [blue, 1NMF.pdb (19)] and dT₇-bound (yellow) Bs-CspB. The five β-strands and their connecting loops are indicated. The r.m.s.d. values of dT₇-bound Bs-CspB of all 67 residues are 0.54 Å (backbone) and 0.94 Å (all heavy atoms). Main deviations between apo and holo form of Bs-CspB are located in loop β1–β2 (L₁₂) and loop β3–β4 (L₃₄).

Figure 4

¹⁵N relaxation and internal motion parameter of free (48) (open symbols) and dT₇-bound (closed symbols) Bs-CspB. (a) heteronuclear NOE hNOE and (c) transversal relaxation rates R₂, as well as (b) order parameters S² and (d) chemical exchange contributions R_ex to R₂. S² and R_ex were obtained from extended Lipari-Szabo analyses of longitudinal relaxation rates R₁ (data not shown), R₂ rates and hNOE using the program MODELFREE (49). Solid and gray bars represent the five β-strands of Bs-CspB.

Figure 5

Nucleic acid-binding site of Bs-CspB determined by NMR spectroscopy and site-directed mutagenesis at (a) front view and (b) back view. The following residues experienced substantial chemical shift changes of their NMR resonances upon binding to dT₇ and are indicated in blue: 7, 10–17, 20, 25, 26–31, 33, 35, 38–42, 54, 56–59 (backbone) and 8, 25, 34, 59, 62 (side chain). Yellow highlighted side chains illustrate aromatic residues, for which the K_D increased above 100 nM after substitution with alanine. For this illustration the NMR structure with the lowest energy was used.

Figure 6

Backbone superposition of the 18 lowest energy NMR structures (yellow) of Bs-CspB/dT₇ and the crystal structure (green) of Bs-CspB/dT₆ (2ES2.pdb). The side chain conformations of the aromatic residues, which facilitate binding of dT₇, are depicted and labeled.