Abstract
The bacterial flagellum is a remarkable molecular motor, whose primary function in bacteria is to facilitate motility through the rotation of a filament protruding from the bacterial cell. A ...cap complex, consisting of an oligomer of the protein FliD, is localized at the tip of the flagellum, and is essential for filament assembly, as well as adherence to surfaces in some bacteria. However, the structure of the intact cap complex, and the molecular basis for its interaction with the filament, remains elusive. Here we report the cryo-EM structure of the
Campylobacter jejuni
cap complex, which reveals that FliD is pentameric, with the N-terminal region of the protomer forming an extensive set of contacts across several subunits, that contribute to FliD oligomerization. We also demonstrate that the native
C. jejuni
flagellum filament is 11-stranded, contrary to a previously published cryo-EM structure, and propose a molecular model for the filament-cap interaction.
Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in ...development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or pH-insensitive mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.
The human ether-à-go-go–related gene (hERG) encodes a K ⁺ channel crucial for repolarization of the cardiac action potential. EAG-related gene (ERG) channels contain a C-terminal cyclic ...nucleotide-binding homology domain coupled to the pore of the channel by a C-linker. Here, we report the structure of the C-linker/cyclic nucleotide-binding homology domain of a mosquito ERG channel at 2.5-Å resolution. The structure reveals that the region expected to form the cyclic nucleotide-binding pocket is negatively charged and is occupied by a short β-strand, referred to as the intrinsic ligand, explaining the lack of direct regulation of ERG channels by cyclic nucleotides. In hERG channels, the intrinsic ligand harbors hereditary mutations associated with long-QT syndrome (LQTS), a potentially lethal cardiac arrhythmia. Mutations in the intrinsic ligand affected hERG channel gating and LQTS mutations abolished hERG currents and altered trafficking of hERG channels, which explains the LQT phenotype. The structure also reveals a dramatically different conformation of the C-linker compared with the structures of the related ether-à-go-go–like K ⁺ and hyperpolarization-activated cyclic nucleotide-modulated channels, suggesting that the C-linker region may be highly dynamic in the KCNH, hyperpolarization-activated cyclic nucleotide-modulated, and cyclic nucleotide-gated channels.
Engineered proteins generally must possess a stable structure in order to achieve their designed function. Stable designs, however, are astronomically rare within the space of all possible amino acid ...sequences. As a consequence, many designs must be tested computationally and experimentally in order to find stable ones, which is expensive in terms of time and resources. Here we use a high-throughput, low-fidelity assay to experimentally evaluate the stability of approximately 200,000 novel proteins. These include a wide range of sequence perturbations, providing a baseline for future work in the field. We build a neural network model that predicts protein stability given only sequences of amino acids, and compare its performance to the assayed values. We also report another network model that is able to generate the amino acid sequences of novel stable proteins given requested secondary sequences. Finally, we show that the predictive model-despite weaknesses including a noisy data set-can be used to substantially increase the stability of both expert-designed and model-generated proteins.
Software to predict the change in protein stability upon point mutation is a valuable tool for a number of biotechnological and scientific problems. To facilitate the development of such software and ...provide easy access to the available experimental data, the ProTherm database was created. Biases in the methods and types of information collected has led to disparity in the types of mutations for which experimental data is available. For example, mutations to alanine are hugely overrepresented whereas those involving charged residues, especially from one charged residue to another, are underrepresented. ProTherm subsets created as benchmark sets that do not account for this often underrepresent tense certain mutational types. This issue introduces systematic biases into previously published protocols' ability to accurately predict the change in folding energy on these classes of mutations. To resolve this issue, we have generated a new benchmark set with these problems corrected. We have then used the benchmark set to test a number of improvements to the point mutation energetics tools in the Rosetta software suite.
Protein stability, assembly, localization and regulation often depend on the formation of disulphide crosslinks between cysteine side chains. Enzymes known as sulphydryl oxidases catalyse de novo ...disulphide formation and initiate intra- and intermolecular dithiol/disulphide relays to deliver the disulphides to substrate proteins. Quiescin sulphydryl oxidase (QSOX) is a unique, multi-domain disulphide catalyst that is localized primarily to the Golgi apparatus and secreted fluids and has attracted attention owing to its overproduction in tumours. In addition to its physiological importance, QSOX is a mechanistically intriguing enzyme, encompassing functions typically carried out by a series of proteins in other disulphide-formation pathways. How disulphides are relayed through the multiple redox-active sites of QSOX and whether there is a functional benefit to concatenating these sites on a single polypeptide are open questions. Here we present the first crystal structure of an intact QSOX enzyme, derived from a trypanosome parasite. Notably, sequential sites in the disulphide relay were found more than 40 Å apart in this structure, too far for direct disulphide transfer. To resolve this puzzle, we trapped and crystallized an intermediate in the disulphide hand-off, which showed a 165° domain rotation relative to the original structure, bringing the two active sites within disulphide-bonding distance. The comparable structure of a mammalian QSOX enzyme, also presented here, shows further biochemical features that facilitate disulphide transfer in metazoan orthologues. Finally, we quantified the contribution of concatenation to QSOX activity, providing general lessons for the understanding of multi-domain enzymes and the design of new catalytic relays.
Biological membranes create compartments, and are usually formed by lipid bilayers. However, in hyperthermophilic archaea that live optimally at temperatures above 80°C the membranes are monolayers ...which resemble fused bilayers. Many double-stranded DNA viruses which parasitize such hosts, including the filamentous virus AFV1 of
, are enveloped with a lipid-containing membrane. Using cryo-EM, we show that the membrane in AFV1 is a ~2 nm-thick monolayer, approximately half the expected membrane thickness, formed by host membrane-derived lipids which adopt a U-shaped 'horseshoe' conformation. We hypothesize that this unusual viral envelope structure results from the extreme curvature of the viral capsid, as 'horseshoe' lipid conformations favor such curvature and host membrane lipids that permit horseshoe conformations are selectively recruited into the viral envelope. The unusual envelope found in AFV1 also has many implications for biotechnology, since this membrane can survive the most aggressive conditions involving extremes of temperature and pH.
Molecular replacement (MR), a method for solving the crystallographic phase problem using phases derived from a model of the target structure, has proven extremely valuable, accounting for the vast ...majority of structures solved by X-ray crystallography. However, when the resolution of data is low, or the starting model is very dissimilar to the target protein, solving structures via molecular replacement may be very challenging. In recent years, protein structure prediction methodology has emerged as a powerful tool in model building and model refinement for difficult molecular replacement problems. This chapter describes some of the tools available in Rosetta for model building and model refinement specifically geared toward difficult molecular replacement cases.
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