Protein Ensemble Generation Through Variational Autoencoder Latent Space Sampling.

Mapping the ensemble of protein conformations that contribute to function and can be targeted by small molecule drugs remains an outstanding challenge. Here, we explore the use of variational autoencoders for reducing the challenge of dimensionality in the protein structure ensemble generation problem. We convert high-dimensional protein structural data into a continuous, low-dimensional representation, carry out a search in this space guided by a structure quality metric, and then use RoseTTAFold guided by the sampled structural information to generate 3D structures. We use this approach to generate ensembles for the cancer relevant protein K-Ras, train the VAE on a subset of the available K-Ras crystal structures and MD simulation snapshots, and assess the extent of sampling close to crystal structures withheld from training. We find that our latent space sampling procedure rapidly generates ensembles with high structural quality and is able to sample within 1 Å of held-out crystal structures, with a consistency higher than that of MD simulation or AlphaFold2 prediction. The sampled structures sufficiently recapitulate the cryptic pockets in the held-out K-Ras structures to allow for small molecule docking.

De novo design of high-affinity binders of bioactive helical peptides.

Many peptide hormones form an α-helix on binding their receptors1-4, and sensitive methods for their detection could contribute to better clinical management of disease5. De novo protein design can now generate binders with high affinity and specificity to structured proteins6,7. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion8 to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar-affinity binders can be generated to helical peptide targets by either refining designs generated with other methods, or completely de novo starting from random noise distributions without any subsequent experimental optimization. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimize by partial diffusion both natural and designed proteins, should be broadly useful.

Biophysical and functional characterization of Norrin signaling through Frizzled4.

Wnt signaling is initiated by Wnt ligand binding to the extracellular ligand binding domain, called the cysteine-rich domain (CRD), of a Frizzled (Fzd) receptor. Norrin, an atypical Fzd ligand, specifically interacts with Fzd4 to activate ß-catenin-dependent canonical Wnt signaling. Much of the molecular basis that confers Norrin selectivity in binding to Fzd4 was revealed through the structural study of the Fzd4CRD-Norrin complex. However, how the ligand interaction, seemingly localized at the CRD, is transmitted across full-length Fzd4 to the cytoplasm remains largely unknown. Here, we show that a flexible linker domain, which connects the CRD to the transmembrane domain, plays an important role in Norrin signaling. The linker domain directly contributes to the high-affinity interaction between Fzd4 and Norrin as shown by ∼10-fold higher binding affinity of Fzd4CRD to Norrin in the presence of the linker. Swapping the Fzd4 linker with the Fzd5 linker resulted in the loss of Norrin signaling, suggesting the importance of the linker in ligand-specific cellular response. In addition, structural dynamics of Fzd4 associated with Norrin binding investigated by hydrogen/deuterium exchange MS revealed Norrin-induced conformational changes on the linker domain and the intracellular loop 3 (ICL3) region of Fzd4. Cell-based functional assays showed that linker deletion, L430A and L433A mutations at ICL3, and C-terminal tail truncation displayed reduced ß-catenin-dependent signaling activity, indicating the functional significance of these sites. Together, our results provide functional and biochemical dissection of Fzd4 in Norrin signaling.