Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion

Abstract

De novo protein design has succeeded in generating a large variety of globular proteins, but the construction of protein scaffolds with cavities that could accommodate large signaling molecules, cofactors, and substrates remains an outstanding challenge. The long, often flexible loops that form such cavities in many natural proteins are difficult to precisely program and thus challenging for computational protein design. Here we describe an alternative approach to this problem. We fused two stable proteins with C2 symmetry—a de novo designed dimeric ferredoxin fold and a de novo designed TIM barrel—such that their symmetry axes are aligned to create scaffolds with large cavities that can serve as binding pockets or enzymatic reaction chambers. The crystal structures of two such designs confirm the presence of a 420 cubic Ångström chamber defined by the top of the designed TIM barrel and the bottom of the ferredoxin dimer. We functionalized the scaffold by installing a metal-binding site consisting of four glutamate residues close to the symmetry axis. The protein binds lanthanide ions with very high affinity as demonstrated by tryptophan-enhanced terbium luminescence. This approach can be extended to other metals and cofactors, making this scaffold a modular platform for the design of binding proteins and biocatalysts.

The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach

“…These data can now be used to accelerate the development of future custom design protein stability curves which, in turn, will expand the biomedical and biotechnological applications of de novo proteins. For example, by fusion to another de novo protein, one of the stabilized scaffolds reported here (DeNovoTIM13) has been successfully used to create a reaction chamber on the top of the barrel, 58 confirming the convenience of working with robust and stable TIM barrels in the path towards functional de novo proteins.…”

Protein Sequence Design with a Learned Potential

“…Sequence overlap between TIM-barrel subunits for model TIM-barrel designs (orange) and previously characterized sequences for the same scaffold (blue), including sTIM-11 (5bvl, S11) [40], DeNovoTIM15 (6wvs, D15) [44], and NovoTIMs (N6, N13, N14a, N14b) [45]. Specific overlaps shown for the region between ( A ) the helices and the barrel, ( B ) the inner part of the barrel, ( C ) exposed regions, and ( D ) all residues.…”

“…( B ) Distribution of rotamer automorphic RMSDs (Å) between crystal structure and template backbone for core and solvent-exposed residues for (top) F2C and (bottom) F15C. ( C ) Sequence overlap between TIM-barrel subunits for model TIM-barrel designs (orange) and previously characterized sequences for the same scaffold (blue), including sTIM-11 (5bvl, S11) [40], DeNovoTIM15 ( 6wvs , D15) [44], and NovoTIMs (N6, N13, N14a, N14b) [45]. Edge thickness increases with overlap.…”

Evolution, folding, and design of TIM barrels and related proteins

“…Structural and folding analysis showed that epistatic effects allow navigating an unexplored region of the stability landscape of natural proteins. One of these DeNovoTIMs was already used in a successful recombination with a de novo designed ferredoxin protein and engineered to bind lanthanide [ 87 ]. In another recent study, Wiese et al extended sTIM11 by successfully incorporating a rationally designed small α-helix into a βα loop [ 88 ].…”