The importance of the location of the N-terminus in successful protein folding in vivo and in vitro.

Protein folding in the cell often begins during translation. Many proteins fold more efficiently cotranslationally than when refolding from a denatured state. Changing the vectorial synthesis of the polypeptide chain through circular permutation could impact functional, soluble protein expression and interactions with cellular proteostasis factors. Here, we measure the solubility and function of every possible circular permutant (CP) of HaloTag in Escherichia coli cell lysate using a gel-based assay, and in living E. coli cells via FACS-seq. We find that 78% of HaloTag CPs retain protein function, though a subset of these proteins are also highly aggregation-prone. We examine the function of each CP in E. coli cells lacking the cotranslational chaperone trigger factor and the intracellular protease Lon and find no significant changes in function as a result of modifying the cellular proteostasis network. Finally, we biophysically characterize two topologically interesting CPs in vitro via circular dichroism and hydrogen-deuterium exchange coupled with mass spectrometry to reveal changes in global stability and folding kinetics with circular permutation. For CP33, we identify a change in the refolding intermediate as compared to wild-type (WT) HaloTag. Finally, we show that the strongest predictor of aggregation-prone expression in cells is the introduction of termini within the refolding intermediate. These results, in addition to our finding that termini insertion within the conformationally restrained core is most disruptive to protein function, indicate that successful folding of circular permutants may depend more on changes in folding pathway and termini insertion in flexible regions than on the availability of proteostasis factors.

Nub1 traps unfolded FAT10 for ubiquitin-independent degradation by the 26S proteasome.

The ubiquitin-like modifier FAT10 targets hundreds of proteins in the mammalian immune system to the 26S proteasome for degradation. This degradation pathway requires the cofactor Nub1, yet the underlying mechanisms remain unknown. Here, we reconstituted a minimal in vitro system and revealed that Nub1 utilizes FAT10's intrinsic instability to trap its N-terminal ubiquitin-like domain in an unfolded state and deliver it to the 26S proteasome for engagement, allowing the degradation of FAT10-ylated substrates in a ubiquitin- and p97-independent manner. Through hydrogen-deuterium exchange, structural modeling, and site-directed mutagenesis, we identified the formation of a peculiar complex with FAT10 that activates Nub1 for docking to the 26S proteasome, and our cryo-EM studies visualized the highly dynamic Nub1 complex bound to the proteasomal Rpn1 subunit during FAT10 delivery and the early stages of ATP-dependent degradation. These studies thus identified a novel mode of cofactor-mediated, ubiquitin-independent substrate delivery to the 26S proteasome that relies on trapping partially unfolded states for engagement by the proteasomal ATPase motor.

The importance of input sequence set to consensus-derived proteins and their relationship to reconstructed ancestral proteins.

A protein sequence encodes its energy landscape-all the accessible conformations, energetics, and dynamics. The evolutionary relationship between sequence and landscape can be probed phylogenetically by compiling a multiple sequence alignment of homologous sequences and generating common ancestors via Ancestral Sequence Reconstruction or a consensus protein containing the most common amino acid at each position. Both ancestral and consensus proteins are often more stable than their extant homologs-questioning the differences between them and suggesting that both approaches serve as general methods to engineer thermostability. We used the Ribonuclease H family to compare these approaches and evaluate how the evolutionary relationship of the input sequences affects the properties of the resulting consensus protein. While the consensus protein derived from our full Ribonuclease H sequence alignment is structured and active, it neither shows properties of a well-folded protein nor has enhanced stability. In contrast, the consensus protein derived from a phylogenetically-restricted set of sequences is significantly more stable and cooperatively folded, suggesting that cooperativity may be encoded by different mechanisms in separate clades and lost when too many diverse clades are combined to generate a consensus protein. To explore this, we compared pairwise covariance scores using a Potts formalism as well as higher-order sequence correlations using singular value decomposition (SVD). We find the SVD coordinates of a stable consensus sequence are close to coordinates of the analogous ancestor sequence and its descendants, whereas the unstable consensus sequences are outliers in SVD space.

Stepwise activation of a metabotropic glutamate receptor.

Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain that is linked via a cysteine-rich domain to their 7-transmembrane domain1. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the extracellular ligand-binding domain to the G protein-coupling 7-transmembrane domain2. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging, we reveal distinct receptor conformations upon allosteric modulator and G protein binding.

Domain coupling in activation of a family C GPCR.

The G protein-coupled metabotropic glutamate receptors form homodimers and heterodimers with highly diverse responses to glutamate and varying physiological function. The molecular basis for this diversity remains poorly delineated. We employ molecular dynamics, single-molecule spectroscopy, and hydrogen-deuterium exchange to dissect the pathway of activation triggered by glutamate. We find that activation entails multiple loosely coupled steps and identify a novel pre-active intermediate whose transition to the active state forms dimer interactions that set signaling efficacy. Such subunit interactions generate functional diversity that differs across homodimers and heterodimers. The agonist-bound receptor is remarkably dynamic, with low occupancy of G protein-coupling conformations, providing considerable headroom for modulation of the landscape by allosteric ligands. Sites of sequence diversity within the dimerization interface and diverse coupling between activation rearrangements may contribute to precise decoding of glutamate signals and transients over broad spatial and temporal scales.

Selection pressures on evolution of ribonuclease H explored with rigorous free-energy-based design.

Understanding natural protein evolution and designing novel proteins are motivating interest in development of high-throughput methods to explore large sequence spaces. In this work, we demonstrate the application of multisite λ dynamics (MSλD), a rigorous free energy simulation method, and chemical denaturation experiments to quantify evolutionary selection pressure from sequence-stability relationships and to address questions of design. This study examines a mesophilic phylogenetic clade of ribonuclease H (RNase H), furthering its extensive characterization in earlier studies, focusing on E. coli RNase H (ecRNH) and a more stable consensus sequence (AncCcons) differing at 15 positions. The stabilities of 32,768 chimeras between these two sequences were computed using the MSλD framework. The most stable and least stable chimeras were predicted and tested along with several other sequences, revealing a designed chimera with approximately the same stability increase as AncCcons, but requiring only half the mutations. Comparing the computed stabilities with experiment for 12 sequences reveals a Pearson correlation of 0.86 and root mean squared error of 1.18 kcal/mol, an unprecedented level of accuracy well beyond less rigorous computational design methods. We then quantified selection pressure using a simple evolutionary model in which sequences are selected according to the Boltzmann factor of their stability. Selection temperatures from 110 to 168 K are estimated in three ways by comparing experimental and computational results to evolutionary models. These estimates indicate selection pressure is high, which has implications for evolutionary dynamics and for the accuracy required for design, and suggests accurate high-throughput computational methods like MSλD may enable more effective protein design.