Guest-protein incorporation into solvent channels of a protein host crystal (hostal)

Abstract

Soaking small molecules into the solvent channels of protein crystals is the most common method of obtaining crystalline complexes with ligands such as substrates or inhibitors. The solvent channels of some protein crystals are large enough to allow the incorporation of macromolecules, but soaking of protein guests into protein crystals has not been reported. Such protein host crystals (here given the name hostals) incorporating guest proteins may be useful for a wide range of applications in biotechnology, for example as cargo systems or for diffraction studies analogous to the crystal sponge method. The present study takes advantage of crystals of the Escherichia coli tryptophan repressor protein (ds-TrpR) that are extensively domain-swapped and suitable for incorporating guest proteins by diffusion, as they are robust and have large solvent channels. Confocal fluorescence microscopy is used to follow the migration of cytochrome c and fluorophore-labeled calmodulin into the solvent channels of ds-TrpR crystals. The guest proteins become uniformly distributed in the crystal within weeks and enriched within the solvent channels. X-ray diffraction studies on host crystals with high concentrations of incorporated guests demonstrate that diffraction limits of ∼2.5 Å can still be achieved. Weak electron density is observed in the solvent channels, but the guest-protein structures could not be determined by conventional crystallographic methods. Additional approaches that increase the ordering of guests in the host crystal are discussed that may support protein structure determination using the hostal system in the future. This host system may also be useful for biotechnological applications where crystallographic order of the guest is not required.

Crystal structures of Val58Ile tryptophan repressor in a domain-swapped array in the presence and absence ofL-tryptophan

“…Thus, whether or not the N-terminal extension is present, the N-terminus points into the channel (Fig. 5), as was observed previously for wildtype ds-TrpR crystals (Lawson et al, 2004;Sprenger et al, 2021). The remainder of the uncleaved Val58Ile ds-TrpR protein structure is essentially identical to that of cleaved Val58Ile ds-TrpR, except for the additional two N-terminal residues.…”

“…These wide channels presumably facilitate diffusion of the ligands l-Trp or 5-bromotryptophan into the crystals to reach the binding site. The channels are also large enough to admit small proteins (horse heart cytochrome c and human calmodulin; Sprenger et al, 2021). The latter property has inspired the investigation of ds-TrpR as a crystalline host to enable the determination of structures of guest proteins that cannot themselves be crystallized, although to date the trial guest proteins cytochrome c and calmodulin have not been sufficiently ordered within the channels for structure solution.…”

“…The fact that the crystallization of TrpR in a domain-swapped form tolerates the presence of a disordered extension of over 20 residues encourages further work to explore a range of N-terminal extensions to use as bait for guest proteins or as a site for fusion of peptides or proteins that cannot otherwise be crystallized. These and other possibilities for using ds-TrpR as a crystalline host system have been discussed in more detail by Sprenger et al (2021) and are being explored in ongoing work.…”

“…During the course of studies to pursue further analysis of Val58Ile TrpR by neutron diffraction based on promising early results (Lawson, 1996a;Daniels et al, 2003), it was discovered that this mutant could be crystallized in the extended domainswapped form under the conditions reported previously for wild-type TrpR. The very large solvent channels typical of ds-TrpRs are being analyzed for possible use as a host system to facilitate diffraction analysis of guest proteins that cannot be crystallized (a so-called hostal system; Sprenger et al, 2021). This system potentially offers a novel approach to the incorporation of biomolecular guests into porous systems, an area of general interest in applied biotechnology today (Yan et al, 2015;Abe & Ueno, 2015;Hartje & Snow, 2018).…”

Time-series analysis of rhenium(I) organometallic covalent binding to a model protein for drug development

“…Solvent channels control access of a compound soaked into a crystal over a period of time from its mother liquor and can be useful to understand interactions between an inhibitor or a substrate with a target protein. This forms an important approach in drug discovery (Sprenger et al, 2021). The natural (i.e.…”