De novo design of protein homodimers containing tunable symmetric protein pockets

Hicks, Derrick R.; Kennedy, Madison; Thompson, Kirsten A.; DeWitt, Michelle; Coventry, Brian; Kang, Alex; Bera, Asim K.; Brunette, TJ; Sankaran, Banumathi; Stoddard, Barry; Baker, David

doi:10.1073/pnas.2113400119

Cited by 13 publications

(16 citation statements)

References 21 publications

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“…The monomer–monomer interfaces and oligomer surfaces were iteratively designed using RosettaScripts 16 , 17 xml scripts following the protocol used for C2 symmetric homo-oligomers. 8 Initially, our design process modified only the interface, as the initial monomers were already assigned a sequence. However, such designs were found to have a high rate of insoluble expression.…”

Section: Resultsmentioning

confidence: 99%

“…A 100 μL aliquot of each sample was then run through a high-performance liquid chromatography system from Agilent using a Superdex 200 10/300 GL column. These fractionation runs were coupled to a Wyatte multiangle light scattering detector in order to determine the absolute molecular weights for each designed protein following the method described in refs ( 8 ) and ( 11 ).…”

Section: Materials and Methodsmentioning

confidence: 99%

“…Previously, a range of pocket sizes and shapes have been designed in monomeric protein systems such as mixed alpha-beta proteins with NTF2-like folds , and beta barrels. , However, the position of the pocket in the center of the monomer restricts the number of positions that can be mutated without disrupting the overall fold geometry. Diverse pocket shapes and sizes have been designed surrounding the central symmetry axis of homodimeric systems, but such pockets are C2 symmetric by construction and are hence not optimal for binding asymmetric substrates and small molecules.…”

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confidence: 99%

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Design of Diverse Asymmetric Pockets in De Novo Homo-oligomeric Proteins

et al. 2023

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A challenge for design of protein–small-molecule recognition is that incorporation of cavities with size, shape, and composition suitable for specific recognition can considerably destabilize protein monomers. This challenge can be overcome through binding pockets formed at homo-oligomeric interfaces between folded monomers. Interfaces surrounding the central homo-oligomer symmetry axes necessarily have the same symmetry and so may not be well suited to binding asymmetric molecules. To enable general recognition of arbitrary asymmetric substrates and small molecules, we developed an approach to designing asymmetric interfaces at off-axis sites on homo-oligomers, analogous to those found in native homo-oligomeric proteins such as glutamine synthetase. We symmetrically dock curved helical repeat proteins such that they form pockets at the asymmetric interface of the oligomer with sizes ranging from several angstroms, appropriate for binding a single ion, to up to more than 20 Å across. Of the 133 proteins tested, 84 had soluble expression in E. coli, 47 had correct oligomeric states in solution, 35 had small-angle X-ray scattering (SAXS) data largely consistent with design models, and 8 had negative-stain electron microscopy (nsEM) 2D class averages showing the structures coming together as designed. Both an X-ray crystal structure and a cryogenic electron microscopy (cryoEM) structure are close to the computational design models. The nature of these proteins as homo-oligomers allows them to be readily built into higher-order structures such as nanocages, and the asymmetric pockets of these structures open rich possibilities for small-molecule binder design free from the constraints associated with monomer destabilization.

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Section: Resultsmentioning

confidence: 99%

Section: Materials and Methodsmentioning

confidence: 99%

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Design of Diverse Asymmetric Pockets in De Novo Homo-oligomeric Proteins

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“…To address the first challenge, we reasoned that a necessary (but not sufficient) criterion for in-phase geometric matching between repeating units on the designed protein and repeating units on the peptide was a correspondence between the superhelices that the two trace out. All repeating polymeric structures trace out superhelices that can be described by three parameters: the translation (rise) along the helical axis per repeat unit; the rotation (twist) around this axis; and the distance (radius) of the repeat unit centroid from the axis 18 , 19 (Fig. 1a ).…”

Section: Design Approachmentioning

confidence: 99%

De novo design of modular peptide-binding proteins by superhelical matching

Bai

Chang

et al. 2023

Nature

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General approaches for designing sequence-specific peptide-binding proteins would have wide utility in proteomics and synthetic biology. However, designing peptide-binding proteins is challenging, as most peptides do not have defined structures in isolation, and hydrogen bonds must be made to the buried polar groups in the peptide backbone1–3. Here, inspired by natural and re-engineered protein–peptide systems4–11, we set out to design proteins made out of repeating units that bind peptides with repeating sequences, with a one-to-one correspondence between the repeat units of the protein and those of the peptide. We use geometric hashing to identify protein backbones and peptide-docking arrangements that are compatible with bidentate hydrogen bonds between the side chains of the protein and the peptide backbone12. The remainder of the protein sequence is then optimized for folding and peptide binding. We design repeat proteins to bind to six different tripeptide-repeat sequences in polyproline II conformations. The proteins are hyperstable and bind to four to six tandem repeats of their tripeptide targets with nanomolar to picomolar affinities in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including ladders of hydrogen bonds from protein side chains to peptide backbones. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating peptide sequences and for disordered regions of native proteins.

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“…C 2 symmetry ensures that the two bound Chl molecules will have near-degenerate site energies, improving the resonance between pigment transitions necessary to create delocalized states (Reppert, 2023). For Chl dimer protein scaffolds, we chose hyperstable C 2 -symmetric repeat protein dimers containing symmetric pockets with tunable sizes and geometries (Brunette et al, 2015(Brunette et al, , 2020Doyle et al, 2015;Fallas et al, 2017;Hicks et al, 2022). In this dimeric repeat protein architecture (Figure 1c), the hydrophobic core is independent from the small molecule binding site, enabling full customization for binding with little impact on the overall protein structure.…”

Section: Introductionmentioning

confidence: 99%

De novo design of energy transfer proteins housing excitonically coupled chlorophyll special pairs

Ennist

Wang

Kennedy

et al. 2023

Preprint

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Natural photosystems couple light harvesting to charge separation using a “special pair” of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independent of complexities of native photosynthetic proteins, and as a first step towards synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that precisely position chlorophyll dimers. X-ray crystallography shows that one designed protein binds two chlorophylls in a binding orientation matching native special pairs, while a second positions them in a previously unseen geometry. Spectroscopy reveals excitonic coupling, and fluorescence lifetime imaging demonstrates energy transfer. We designed special pair proteins to assemble into 24-chlorophyll octahedral nanocages; the design model and cryo-EM structure are nearly identical. The design accuracy and energy transfer function of these special pair proteins suggest that de novo design of artificial photosynthetic systems is within reach of current computational methods.

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De novo design of protein homodimers containing tunable symmetric protein pockets

Cited by 13 publications

References 21 publications

Design of Diverse Asymmetric Pockets in De Novo Homo-oligomeric Proteins

Design of Diverse Asymmetric Pockets in De Novo Homo-oligomeric Proteins

De novo design of modular peptide-binding proteins by superhelical matching

De novo design of energy transfer proteins housing excitonically coupled chlorophyll special pairs

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