Modular Protein-DNA Cocrystals as Precise, Programmable Assembly Scaffolds

Abstract

High-precision nanomaterials to entrap DNA-binding molecules are sought after for applications such as controlled drug delivery and scaffold-assisted structural biology. Here, we engineered protein-DNA cocrystals to serve as scaffolds for DNA-binding molecules. The designed cocrystals, isoreticular cocrystals, contain DNA-binding protein and cognate DNA blocks where the DNA-DNA junctions stack end-to-end. Furthermore, the crystal symmetry allows topology preserving (isoreticular) expansion of the DNA stack without breaking protein-protein contacts, hence providing larger solvent channels for guest diffusion. Experimentally, the resulting designed isoreticular cocrystal adopted an interpenetrating I222 lattice, a phenomenon previously observed in metal-organic frameworks (MOFs). The interpenetrating lattice crystallized dependably in the same space group despite myriad modifications at the DNA-DNA junctions. Assembly was modular with respect to the DNA inserted for expansion, providing an interchangeable DNA sequence for guest-specified scaffolding. Also, the DNA-DNA junctions were tunable, accommodating varied sticky base overhang lengths and terminal phosphorylation. As a proof of concept, we used the interpenetrating scaffold crystals to separately entrap three distinct guest molecules during crystallization. Isoreticular cocrystal design offers a route to a programmable scaffold for DNA-binding molecules, and the design principles may be applied to existing cocrystals to develop scaffolding materials.

Keywords: DNA-binding protein; X-ray crystallography; cocrystal; design; scaffold.

Figure 1

An isoreticular cocrystal (A) is built of a scaffold protein (gray sphere) and DNA (green rod). Inserted DNA (dark green rod) might be added at the DNA–DNA junctions, and insertion lengths that respect the DNA helical twist may allow junctions to be stabilized by coding sticky overhangs. (B) The simplest example of crystal expansion would occur in a 1D expansion with parallel DNA stacks throughout the crystal. (C) 2D expansion may occur when DNA–DNA junctions mediate growth in two dimensions. (D) The inserted DNA provides site-specific scaffolding for the DNA-binding guest of interest.

Figure 2

Asymmetric unit of cocrystal 1 (A) is the complex of RepE54 replication initiator protein and a cognate 21-mer binding sequence (based on PDB code 7rva). The CC1 lattice (B) is in a C121 space group, and there are two protein–protein interfaces (C) conserved throughout the lattice. Upon expansion of the DNA with 10 additional base pairs (D) the lattice is highly porous (E) and the DNA–DNA junctions (G) are accessible for DNA-binding guest targets and are not sequence constrained by scaffold interactions.

Figure 3

Target porous lattice (A), shown here in the I121 setting, was similar to (B) half of the contents of the I222 experimental structure. (C) The other half of the I222 experimental structure is obtained via a 180° rotation about the x-axis (vertical in this diagram). (D) The full I222 structure requires close packing of DNA duplexes and involves additional 2-fold symmetry axes perpendicular to the y-axis (which is itself perpendicular to the page).

Figure 4

Isoreticular cocrystal design schemes vary the original, nonexpanded duplex (A). The symmetrical expansion (B) fused 5 base pairs to each side of the original duplex. The asymmetrical expansion (C) fused 10 base pairs to a single side of the original duplex. The scaffold-insert expansion (D) maintained a 21 base pair scaffold strand and added an independent 10 base pair insert strand with matching sticky overhangs to the scaffold strand. In each expansion scheme, the DNA–DNA junctions were tunable with varied sticky base overhang lengths and terminal phosphorylation (no phosphate, 3′P, or 5′P).

Figure 5

Interpenetrating CC1^+10bp crystal growth for various DNA blocks cocrystallized with RepE54 Transcription Factor. Symmetric expansion crystals were grown with (A) G-C rich or (B) T-A rich addition. (C) Asymmetric expansion crystals were grown with a G-C rich addition. Scaffold-insert expansion crystals were grown with a (D) G-C rich insert strand or (E) T-A rich insert strand. (F–J) Symmetric expansion crystals grew with varied sticky base overhang lengths: blunt end, 1-nt, 2-nt, 3-nt, and 4-nt. Note: panel H intentionally duplicates panel A. Matching crystal growth conditions are found in Table S1. PDB codes for A–E are in the upper right corner. Scale bars are 100 μm.

Figure 6

Symmetric expanded CC1^+10bp was designed with the Engrailed homeodomain (EnH) binding site (A). After cocrystallization with EnH-eGFP fusion protein (B), the crystal fluoresced when exposed to 488 nm with confocal microscopy, indicating guest EnH-eGFP entrapment (C–D). The control crystal, grown without EnH-eGFP, did not fluoresce. Scale bar: 100 μm.

Figure 7

(A) The scaffold-insert expanded complex shows the scaffold DNA in cyan and the insert strand in dark blue. The TAMRA-labeled thymine 2 (magenta) is conjugated on the C7 atom (B). Cocrystals grew with the TAMRA-T2 in the interpenetrating I222 space group. Crystals consistently grew for both a G-C rich insert sequence (C) and a T-A rich insert sequence (D). Substituents R₁ and R₂ are the 5′ and 3′ neighboring nucleobase, respectively.

Figure 8

X-ray diffraction highlight of netropsin bound to CC1^+10bp. (A) The electron density indicated that netropsin, shown in magenta, was bound at the 5′-AATT site. The cocrystal resolution was 3.1 Å. (B) The Polder map (an omit map for netropsin that excludes bulk solvent generated with PHENIX Polder Maps) in gray mesh shows the electron density around netropsin in the binding pocket. The DNA backbone of the binding site is highlighted in orange. (C) The Polder map of netropsin (contour level 3 rmsd).