Simple yet functional phosphate-loop proteins

Abstract

Abundant and essential motifs, such as phosphate-binding loops (P-loops), are presumed to be the seeds of modern enzymes. The Walker-A P-loop is absolutely essential in modern NTPase enzymes, in mediating binding, and transfer of the terminal phosphate groups of NTPs. However, NTPase function depends on many additional active-site residues placed throughout the protein's scaffold. Can motifs such as P-loops confer function in a simpler context? We applied a phylogenetic analysis that yielded a sequence logo of the putative ancestral Walker-A P-loop element: a β-strand connected to an α-helix via the P-loop. Computational design incorporated this element into de novo designed β-α repeat proteins with relatively few sequence modifications. We obtained soluble, stable proteins that unlike modern P-loop NTPases bound ATP in a magnesium-independent manner. Foremost, these simple P-loop proteins avidly bound polynucleotides, RNA, and single-strand DNA, and mutations in the P-loop's key residues abolished binding. Binding appears to be facilitated by the structural plasticity of these proteins, including quaternary structure polymorphism that promotes a combined action of multiple P-loops. Accordingly, oligomerization enabled a 55-aa protein carrying a single P-loop to confer avid polynucleotide binding. Overall, our results show that the P-loop Walker-A motif can be implemented in small and simple β-α repeat proteins, primarily as a polynucleotide binding motif.

Keywords: RNA binding protein; Walker-A; conformational diversity; de novo protein design; protein evolution.

Fig. 1.

(A) Structural alignment of β-(P-loop)-α motifs of different P-loop NTPase (the canonical Walker-A residues: G₁, G₂, and G₃ are in pink, K₄ in black, and [T/S]₅in white; G1-XX-G2-X-G3-K4-(S/T)5). (B) Sequence logo representing the inferred ancestral β-(P-loop)-α profile. (C) Sequence alignment of the most probable ancestral β-(P-loop)-α sequence and of the N-terminal segments of the PLoop designs. (D) Schematic representation of the secondary structure and topology of the PLoop designs. Helices are represented by rectangles or circles, strands by arrows or triangles, and the P-loops by pink segments. The flanking strand of the β-(P-loop)-α segment is in blue and the helix in red.

Fig. 2.

Structural characteristics of C-PLoop. (A) CD spectra demonstrate high thermostability. (B) Native MS analysis indicating monomers alongside a minor fraction of dimers. Noted are the measured MWs (the expected MW is 12,474 Da). (C) Two-dimensional ¹H¹⁵N-HSQC spectra at 25 °C. (D) Structural models derived from NMR data. (Upper) The observed major C-PLoop conformation. (Lower) Alignment of the β-(P-loop)-α elements of the two identified conformations (major in blue, PDB ID code 6C2U, and minor in orange, PDB ID code 6C2V). RMSD values: 1.48 Å between the observed major conformation and the designed model, and 2.1 Å for the minor conformation.

Fig. 3.

Polynucleotide and ATP binding properties of A- to D-PLoops. (A) Binding to 287-nt ssDNA and the corresponding dsDNA, determined by ELISA. Black circles mark the 2N3Z control that showed no binding. (B) ELISA at 0.2 μM (plain bars) and 1 μM (striated bars) protein concentrations with immobilized homomeric ssDNA (black circles mark the 2N3Z control). (C) MST profiles with fluorescently labeled dC₁₅. The lines designate a fit to a sigmoidal binding curve. (D) SPR sensograms at 5-μM protein concentration on ssDNA, RNA, and ATP. (Right) Maximal RU values at different protein concentrations. (E) Effect of the His-tag on C-PLoop’s binding properties. The His-tag may trigger formation of high oligomeric forms upon binding causing a slow and variable association phase (SI Appendix, Fig. S6). SPR sensograms at 5-μM protein concentration over several immobilized biotinylated ligands. (Right) Maximal RU values at different protein concentration.

Fig. 4.

P-loop residues mediate ligand binding. (A) Schematic representation of the Walker-A motif. (B) Binding of B-PLoop and its mutants to ssDNA, RNA, and ATP, assayed by SPR. Shown are the initial slopes derived from plots of maximal RU versus protein concentration (as in Fig. 3D). (C) MST profiles of the titration of dC₁₅ with B-PLoop and its double mutant. The lines represent a fit to sigmoidal binding curves.

Fig. 5.

C-PLoop mediates ATP hydrolysis. (A) ³¹P-NMR of 0.5 mM ATP in 20 mM Mes, 50 mM NaCl, pH 6 at room temperature (Top); the same solution with 1 mM C-PLoop (Middle) or C-PLoop-G3E/K4Q (Bottom). The C-PLoop constructs were purified by His-tag chromatography, anion exchange, and gel filtration. (B) The rates of ATP, ADP, and AMP hydrolysis upon incubation with 62.5 μM C-PLoop or C-PLoop–G3E/K4Q, or with no protein. Rates were measured at at 45 °C, pH 6.0; the lines represent the fit to a single exponential and the apparent rate constants are annotated. (C) Addition of either 1 mM MgCl₂ or of 1 mM EDTA did not affect ATP hydrolysis by C-PLoop (rates measured at 45 °C, pH 6.0, after 240 min, with 80 μM protein). (D) SPR sensograms of two C-PLoop preparations (with immobilized dG₁₅, as in Fig. 3D). Shown in green are injections of 0.125, 0.25, 0.5, 1, 2, and 5 μM protein. The inactive C-PLoop–G3E/K4Q mutant at 5 μM is shown in red (the close to baseline traces indicate no binding).

Fig. 6.

Half–B-PLoop and its mutants. (A) Structural cartoon of the 55-aa half–B-PLoop (grafted from the Rosetta design model of the intact protein). The P-loop Walker-A motif is in blue. (B) SDS/PAGE of intact 2N3Z, B- and C-PLoop, and of their corresponding halves. (C) ELISA at 0.1-μM protein concentration with ssDNA (black circles mark the half-2N3Z control). (D) SPR sensograms of half–B-PLoop and it mutants at 5-μM protein concentration on ssDNA. (Right) Maximal RU values at different protein concentrations, including of the control half-2N3Z.