Comprehensive structure and functional adaptations of the yeast nuclear pore complex.

Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.

Structural basis of mitochondrial protein import by the TIM23 complex.

Mitochondria import nearly all of their approximately 1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope1-5. Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM23 complex, mediates import of presequence-containing proteins (preproteins) into the mitochondrial matrix and inner membrane. Among about ten different subunits of the TIM23 complex, the essential multipass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel6-11. However, the mechanism by which these subunits form a translocation path in the membrane and enable the import process remains unclear due to a lack of structural information. Here we determined the cryo-electron microscopy structure of the core TIM23 complex (heterotrimeric Tim17-Tim23-Tim44) from Saccharomyces cerevisiae. Contrary to the prevailing model, Tim23 and Tim17 themselves do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Our structural and biochemical analyses show that the cavity of Tim17, but not Tim23, forms the protein translocation path, whereas Tim23 probably has a structural role. The results further suggest that, during translocation of substrate polypeptides, the nonessential subunit Mgr2 seals the lateral opening of the Tim17 cavity to facilitate the translocation process. We propose a new model for the TIM23-mediated protein import and sorting mechanism, a central pathway in mitochondrial biogenesis.

Weak tension accelerates hybridization and dehybridization of short oligonucleotides.

The hybridization and dehybridization of DNA subject to tension is relevant to fundamental genetic processes and to the design of DNA-based mechanobiology assays. While strong tension accelerates DNA melting and decelerates DNA annealing, the effects of tension weaker than 5 pN are less clear. In this study, we developed a DNA bow assay, which uses the bending rigidity of double-stranded DNA (dsDNA) to exert weak tension on a single-stranded DNA (ssDNA) target in the range of 2-6 pN. Combining this assay with single-molecule FRET, we measured the hybridization and dehybridization kinetics between a 15 nt ssDNA under tension and a 8-9 nt oligonucleotide, and found that both the hybridization and dehybridization rates monotonically increase with tension for various nucleotide sequences tested. These findings suggest that the nucleated duplex in its transition state is more extended than the pure dsDNA or ssDNA counterpart. Based on coarse-grained oxDNA simulations, we propose that this increased extension of the transition state is due to steric repulsion between the unpaired ssDNA segments in close proximity to one another. Using linear force-extension relations verified by simulations of short DNA segments, we derived analytical equations for force-to-rate conversion that are in good agreement with our measurements.

Proton transfer activity of the reconstituted Mycobacterium tuberculosis MmpL3 is modulated by substrate mimics and inhibitors.

Transporters belonging to the Resistance-Nodulation-cell Division (RND) superfamily of proteins such as Mycobacterium tuberculosis MmpL3 and its analogs are the focus of intense investigations due to their importance in the physiology of Corynebacterium-Mycobacterium-Nocardia species and antimycobacterial drug discovery. These transporters deliver trehalose monomycolates, the precursors of major lipids of the outer membrane, to the periplasm by a proton motive force-dependent mechanism. In this study, we successfully purified, from native membranes, the full-length and the C-terminal truncated M. tuberculosis MmpL3 and Corynebacterium glutamicum CmpL1 proteins and reconstituted them into proteoliposomes. We also generated a series of substrate mimics and inhibitors specific to these transporters, analyzed their activities in the reconstituted proteoliposomes, and carried out molecular dynamics simulations of the model MmpL3 transporter at different pH. We found that all reconstituted proteins facilitate proton translocation across a phospholipid bilayer, but MmpL3 and CmpL1 differ dramatically in their responses to pH and interactions with substrate mimics and indole-2-carboxamide inhibitors. Our results further suggest that some inhibitors abolish the transport activity of MmpL3 and CmpL1 by inhibition of proton translocation.

Generative ß-hairpin design using a residue-based physicochemical property landscape.

De novo peptide design is a new frontier that has broad application potential in the biological and biomedical fields. Most existing models for de novo peptide design are largely based on sequence homology that can be restricted based on evolutionarily derived protein sequences and lack the physicochemical context essential in protein folding. Generative machine learning for de novo peptide design is a promising way to synthesize theoretical data that are based on, but unique from, the observable universe. In this study, we created and tested a custom peptide generative adversarial network intended to design peptide sequences that can fold into the ß-hairpin secondary structure. This deep neural network model is designed to establish a preliminary foundation of the generative approach based on physicochemical and conformational properties of 20 canonical amino acids, for example, hydrophobicity and residue volume, using extant structure-specific sequence data from the PDB. The beta generative adversarial network model robustly distinguishes secondary structures of ß hairpin from α helix and intrinsically disordered peptides with an accuracy of up to 96% and generates artificial ß-hairpin peptide sequences with minimum sequence identities around 31% and 50% when compared against the current NCBI PDB and nonredundant databases, respectively. These results highlight the potential of generative models specifically anchored by physicochemical and conformational property features of amino acids to expand the sequence-to-structure landscape of proteins beyond evolutionary limits.

SANS reveals lipid-dependent oligomerization of an intramembrane aspartyl protease from H. volcanii.

Reactions that occur within the lipid membrane involve, at minimum, ternary complexes among the enzyme, substrate, and lipid. For many systems, the impact of the lipid in regulating activity or oligomerization state is poorly understood. Here, we used small-angle neutron scattering (SANS) to structurally characterize an intramembrane aspartyl protease (IAP), a class of membrane-bound enzymes that use membrane-embedded aspartate residues to hydrolyze transmembrane segments of biologically relevant substrates. We focused on an IAP ortholog from the halophilic archaeon Haloferax volcanii (HvoIAP). HvoIAP purified in n-dodecyl-ß-D-maltoside (DDM) fractionates on size-exclusion chromatography (SEC) as two fractions. We show that, in DDM, the smaller SEC fraction is consistent with a compact HvoIAP monomer. Molecular dynamics flexible fitting conducted on an AlphaFold2-generated monomer produces a model in which loops are compact alongside the membrane-embedded helices. In contrast, SANS data collected on the second SEC fraction indicate an oligomer consistent with an elongated assembly of discrete HvoIAP monomers. Analysis of in-line SEC-SANS data of the HvoIAP oligomer, the first such experiment to be conducted on a membrane protein at Oak Ridge National Lab (ORNL), shows a diversity of elongated and spherical species, including one consistent with the tetrameric assembly reported for the Methanoculleus marisnigri JR1 IAP crystal structure not observed previously in solution. Reconstitution of monomeric HvoIAP into bicelles increases enzyme activity and results in the assembly of HvoIAP into a species with similar dimensions as the ensemble of oligomers isolated from DDM. Our study reveals lipid-mediated HvoIAP self-assembly and demonstrates the utility of in-line SEC-SANS in elucidating oligomerization states of small membrane proteins.

Species-agnostic polymeric formulations for inhalable messenger RNA delivery to the lung.

Messenger RNA has now been used to vaccinate millions of people. However, the diversity of pulmonary pathologies, including infections, genetic disorders, asthma and others, reveals the lung as an important organ to directly target for future RNA therapeutics and preventatives. Here we report the screening of 166 polymeric nanoparticle formulations for functional delivery to the lungs, obtained from a combinatorial synthesis approach combined with a low-dead-volume nose-only inhalation system for mice. We identify P76, a poly-ß-amino-thio-ester polymer, that exhibits increased expression over formulations lacking the thiol component, delivery to different animal species with varying RNA cargos and low toxicity. P76 allows for dose sparing when delivering an mRNA-expressed Cas13a-mediated treatment in a SARS-CoV-2 challenge model, resulting in similar efficacy to a 20-fold higher dose of a neutralizing antibody. Overall, the combinatorial synthesis approach allowed for the discovery of promising polymeric formulations for future RNA pharmaceutical development for the lungs.

Broadening access to small-molecule parameterization with the force field toolkit.

Access to accurate force-field parameters for small molecules is crucial for computational studies of their interactions with proteins. Although a number of general force fields for small molecules exist, e.g., CGenFF, GAFF, and OPLS, they do not cover all common chemical groups and their combinations. The Force Field Toolkit (ffTK) provides a comprehensive graphical interface that streamlines the development of classical parameters for small molecules directly from quantum mechanical (QM) calculations, allowing for force-field generation for almost any chemical group and validation of the fit relative to the target data. ffTK relies on supported external software for the QM calculations, but it can generate the necessary QM input files and parse and analyze the QM output. In previous ffTK versions, support for Gaussian and ORCA QM packages was implemented. Here, we add support for Psi4, an open-source QM package free for all users, thereby broadening user access to ffTK. We also compare the parameter sets obtained with the new ffTK version using Gaussian, ORCA, and Psi4 for three molecules: pyrrolidine, n-propylammonium cation, and chlorobenzene. Despite minor differences between the resulting parameter sets for each compound, most prominently in the dihedral and improper terms, we show that conformational distributions sampled in molecular dynamics simulations using these parameter sets are quite comparable.

Uncovering the folding mechanism of pertactin: A comparative study of isolated and vectorial folding.

Autotransporters are a large family of virulence factors found in Gram-negative bacteria that play important roles in their pathogenesis. The passenger domain of autotransporters is almost always composed of a large ß-helix, with only a small portion of it being relevant to its virulence function. This has led to the hypothesis that the folding of the ß-helical structure aids the secretion of the passenger domain across the Gram-negative outer membrane. In this study, we used molecular dynamics simulations and enhanced sampling methods to investigate the stability and folding of the passenger domain of pertactin, an autotransporter from Bordetella pertussis. Specifically, we employed steered molecular dynamics to simulate the unfolding of the entire passenger domain as well as self-learning adaptive umbrella sampling to compare the energetics of folding rungs of the ß-helix independently ("isolated folding") versus folding rungs on top of a previously folded rung ("vectorial folding"). Our results showed that vectorial folding is highly favorable compared with isolated folding; moreover, our simulations showed that the C-terminal rung of the ß-helix is the most resistant to unfolding, in agreement with previous studies that found the C-terminal half of the passenger domain to be more stable than the N-terminal one. Overall, this study provides new insights into the folding process of an autotransporter passenger domain and its potential role in secretion across the outer membrane.

Insights into substrate transport and water permeation in the mycobacterial transporter MmpL3.

Mycobacteria, such as Mycobacterium tuberculosis, are characterized by a uniquely thick and waxy cell envelope that consists of two membranes, with a variety of mycolates comprising their outer membrane (OM). The protein Mycobacterial membrane protein Large 3 (MmpL3) is responsible for the transport of a primary OM component, trehalose monomycolate (TMM), from the inner (cytoplasmic) membrane (IM) to the periplasmic space, a process driven by the proton gradient. Although multiple structures of MmpL3 with bound substrates have been solved, the exact pathway(s) for TMM or proton transport remains elusive. Here, employing molecular dynamics simulations we investigate putative pathways for either transport species. We hypothesized that MmpL3 will cycle through similar conformational states as the related transporter AcrB, which we used as targets for modeling the conformation of MmpL3. A continuous water pathway through the transmembrane region was found in one of these states, illustrating a putative pathway for protons. Additional equilibrium simulations revealed that TMM can diffuse from the membrane into a binding pocket in MmpL3 spontaneously. We also found that acetylation of TMM, which is required for transport, makes it more stable within MmpL3's periplasmic cavity compared with the unacetylated form.

Modeling intermediates of BamA folding an outer membrane protein.

BamA, the core component of the ß-barrel assembly machinery complex, is an integral outer-membrane protein (OMP) in Gram-negative bacteria that catalyzes the folding and insertion of OMPs. A key feature of BamA relevant to its function is a lateral gate between its first and last ß-strands. Opening of this lateral gate is one of the first steps in the asymmetric-hybrid-barrel model of BamA function. In this study, multiple hybrid-barrel folding intermediates of BamA and a substrate OMP, EspP, were constructed and simulated to better understand the model's physical consequences. The hybrid-barrel intermediates consisted of the BamA ß-barrel and its POTRA5 domain and either one, two, three, four, five, or six ß-hairpins of EspP. The simulation results support an asymmetric-hybrid-barrel model in which the BamA N-terminal ß-strand forms stronger interactions with the substrate OMP than the C-terminal ß-strand. A consistent "B"-shaped conformation of the final folding intermediate was observed, and the shape of the substrate ß-barrel within the hybrid matched the shape of the fully folded substrate. Upon further investigation, inward-facing glycines were found at sharp bends within the hybrid and fully folded ß-barrels. Together, the data suggest an influence of sequence on shape of the substrate barrel throughout the OMP folding process and of the fully folded OMP.

Resolving the Hydride Transfer Pathway in Oxidative Conversion of Proline to Pyrrole.

Thiotemplated pyrrole is a prevailing intermediate in the synthesis of numerous natural products in which the pyrrole is tethered to a carrier protein (CP). Biosynthesis of the pyrrole requires oxidation of an l-proline side chain. Herein, we investigate the biocatalytic mechanism of proline-to-pyrrole synthesis by molecular dynamics simulations, quantum mechanics/molecular mechanics simulations, and electronic structure calculations using the recently reported (Thapa, H. R., et al. Biochemistry 2019, 58, 918) structure of a type II nonribosomal protein synthetase (NRPS) Bmp3-Bmp1 (Oxidase-CP) complex. The substrate (l-proline) is attached to the Bmp1(CP), and the catalytic site is located inside the flavin-dependent oxidase (Bmp3). We show that the FAD isoalloxazine ring is stabilized in the catalytic site of Bmp3 by strong hydrogen bonding with Asn123, Ile125, Ser126, and Thr158. After the initial deprotonation followed by an enamine-imine tautomerization, oxidation of the C2-C3 or C2-N1 bond, through a hydride transfer (from either C3 or N1), is required for the pyrrole synthesis. Computational results indicate that the hydride transfer is more likely to occur from C3 than N1. Additionally, we demonstrate the elasticity in the oxidase active site through enzymatic synthesis of proline derivatives.

Point Mutations in TbpA Abrogate Human Transferrin Binding in Neisseria gonorrhoeae.

TonB-dependent transporters (TDTs) are essential proteins for metal acquisition, an important step in the growth and pathogenesis of many pathogens, including Neisseria gonorrhoeae, the causative agent of gonorrhea. There is currently no available vaccine for gonorrhea; TDTs are being investigated as vaccine candidates because they are highly conserved and expressed in vivo. Transferrin binding protein A (TbpA) is an essential virulence factor in the initiation of experimental infection in human males and functions by acquiring iron upon binding to host transferrin (human transferrin [hTf]). The loop 3 helix (L3H) is a helix finger that inserts into the hTf C-lobe and is required for hTf binding and subsequent iron acquisition. This study identified and characterized the first TbpA single-point substitutions resulting in significantly decreased hTf binding and iron acquisition, suggesting that the helix structure is more important than charge for hTf binding and utilization. The tbpA D355P ΔtbpB and tbpA A356P ΔtbpB mutants demonstrated significantly reduced hTf binding and impaired iron uptake from Fe-loaded hTf; however, only the tbpA A356P ΔtbpB mutant was able to grow when hTf was the sole source of iron. The expression of tbpB was able to restore function in all tbpA mutants. These results implicate both D355 and A356 in the key binding, extraction, and uptake functions of gonococcal TbpA.

Joint neutron/molecular dynamics vibrational spectroscopy reveals softening of HIV-1 protease upon binding of a tight inhibitor.

Biomacromolecules are inherently dynamic, and their dynamics are interwoven into function. The fast collective vibrational dynamics in proteins occurs in the low picosecond timescale corresponding to frequencies of ∼5-50 cm-1. This sub-to-low THz frequency regime covers the low-amplitude collective breathing motions of a whole protein and vibrations of the constituent secondary structure elements, such as α-helices, ß-sheets and loops. We have used inelastic neutron scattering experiments in combination with molecular dynamics simulations to demonstrate the vibrational dynamics softening of HIV-1 protease, a target of HIV/AIDS antivirals, upon binding of a tight clinical inhibitor darunavir. Changes in the vibrational density of states of matching structural elements in the two monomers of the homodimeric protein are not identical, indicating asymmetric effects of darunavir on the vibrational dynamics. Three of the 11 major secondary structure elements contribute over 40% to the overall changes in the vibrational density of states upon darunavir binding. Molecular dynamics simulations informed by experiments allowed us to estimate that the altered vibrational dynamics of the protease would contribute -3.6 kcal mol-1 at 300 K, or 25%, to the free energy of darunavir binding. As HIV-1 protease drug resistance remains a concern, our results open a new avenue to help establish a direct quantitative link between protein vibrational dynamics and drug resistance.

Bifunctional Janus Particles as Multivalent Synthetic Nanoparticle Antibodies (SNAbs) for Selective Depletion of Target Cells.

Monoclonal antibodies (mAb) have had a transformative impact on treating cancers and immune disorders. However, their use is limited by high development time and monetary cost, manufacturing complexities, suboptimal pharmacokinetics, and availability of disease-specific targets. To address some of these challenges, we developed an entirely synthetic, multivalent, Janus nanotherapeutic platform, called Synthetic Nanoparticle Antibodies (SNAbs). SNAbs, with phage-display-identified cell-targeting ligands on one "face" and Fc-mimicking ligands on the opposite "face", were synthesized using a custom, multistep, solid-phase chemistry method. SNAbs efficiently targeted and depleted myeloid-derived immune-suppressor cells (MDSCs) from mouse-tumor and rat-trauma models, ex vivo. Systemic injection of MDSC-targeting SNAbs efficiently depleted circulating MDSCs in a mouse triple-negative breast cancer model, enabling enhanced T cell and Natural Killer cell infiltration into tumors. Our results demonstrate that SNAbs are a versatile and effective functional alternative to mAbs, with advantages of a plug-and-play, cell-free manufacturing process, and high-throughput screening (HTS)-enabled library of potential targeting ligands.

Lpp positions peptidoglycan at the AcrA-TolC interface in the AcrAB-TolC multidrug efflux pump.

The multidrug efflux pumps of Gram-negative bacteria are a class of complexes that span the periplasm, coupling both the inner and outer membranes to expel toxic molecules. The best-characterized example of these tripartite pumps is the AcrAB-TolC complex of Escherichia coli. However, how the complex interacts with the peptidoglycan (PG) cell wall, which is anchored to the outer membrane (OM) by Braun's lipoprotein (Lpp), is still largely unknown. In this work, we present molecular dynamics simulations of a complete, atomistic model of the AcrAB-TolC complex with the inner membrane, OM, and PG layers all present. We find that the PG localizes to the junction of AcrA and TolC, in agreement with recent cryo-tomography data. Free-energy calculations reveal that the positioning of PG is determined by the length and conformation of multiple Lpp copies anchoring it to the OM. The distance between the PG and OM measured in cryo-electron microscopy images of wild-type E. coli also agrees with the simulation-derived spacing. Sequence analysis of AcrA suggests a conserved role for interactions with PG in the assembly and stabilization of efflux pumps, one that may extend to other trans-envelope complexes as well.

Gatekeeping Ketosynthases Dictate Initiation of Assembly Line Biosynthesis of Pyrrolic Polyketides.

Assembly line biosynthesis of polyketide natural products involves checkpoints where identities of thiotemplated intermediates are verified before polyketide extension reactions are allowed to proceed. Determining what these checkpoints are and how they operate is critical for reprogramming polyketide assembly lines. Here we demonstrate that ketosynthase (KS) domains can perform this gatekeeping role. By comparing the substrate specificities for polyketide synthases that extend pyrrolyl and halogenated pyrrolyl substrates, we find that KS domains that need to differentiate between these two substrates exercise high selectivity. We additionally find that amino acid residues in the KS active site facilitate this selectivity and that these residues are amenable to rational engineering. On the other hand, KS domains that do not need to make selectivity decisions in their native physiological context are substrate-promiscuous. We also provide evidence that delivery of substrates to polyketide synthases by non-native carrier proteins is accompanied by reduced biosynthetic efficiency.

Membrane thinning and lateral gating are consistent features of BamA across multiple species.

In Gram-negative bacteria, the folding and insertion of ß-barrel outer membrane proteins (OMPs) to the outer membrane are mediated by the ß-barrel assembly machinery (BAM) complex. Two leading models of this process have been put forth: the hybrid barrel model, which claims that a lateral gate in BamA's ß-barrel can serve as a template for incoming OMPs, and the passive model, which claims that a thinned membrane near the lateral gate of BamA accelerates spontaneous OMP insertion. To examine the key elements of these two models, we have carried out 45.5 µs of equilibrium molecular dynamics simulations of BamA with and without POTRA domains from Escherichia coli, Salmonella enterica, Haemophilus ducreyi and Neisseria gonorrhoeae, together with BamA's homolog, TamA from E. coli, in their native, species-specific outer membranes. In these equilibrium simulations, we consistently observe membrane thinning near the lateral gate for all proteins. We also see occasional spontaneous lateral gate opening and sliding of the ß-strands at the gate interface for N. gonorrhoeae, indicating that the gate is dynamic. An additional 14 µs of free-energy calculations shows that the energy necessary to open the lateral gate in BamA/TamA varies by species, but is always lower than the Omp85 homolog, FhaC. Our combined results suggest OMP insertion utilizes aspects of both the hybrid barrel and passive models.

C-terminal kink formation is required for lateral gating in BamA.

In Gram-negative bacteria, the outer membrane contains primarily ß-barrel transmembrane proteins and lipoproteins. The insertion and assembly of ß-barrel outer-membrane proteins (OMPs) is mediated by the ß-barrel assembly machinery (BAM) complex, the core component of which is the 16-stranded transmembrane ß-barrel BamA. Recent studies have indicated a possible role played by the seam between the first and last ß-barrel strands of BamA in the OMP insertion process through lateral gating and a destabilized membrane region. In this study, we have determined the stability and dynamics of the lateral gate through over 12.5 µs of equilibrium simulations and 4 µs of free-energy calculations. From the equilibrium simulations, we have identified a persistent kink in the C-terminal strand and observed spontaneous lateral-gate separation in a mimic of the native bacterial outer membrane. Free-energy calculations of lateral gate opening revealed a significantly lower barrier to opening in the C-terminal kinked conformation; mutagenesis experiments confirm the relevance of C-terminal kinking to BamA structure and function.

Folding and Insertion of Transmembrane Helices at the ER.

In eukaryotic cells, the endoplasmic reticulum (ER) is the entry point for newly synthesized proteins that are subsequently distributed to organelles of the endomembrane system. Some of these proteins are completely translocated into the lumen of the ER while others integrate stretches of amino acids into the greasy 30 Å wide interior of the ER membrane bilayer. It is generally accepted that to exist in this non-aqueous environment the majority of membrane integrated amino acids are primarily non-polar/hydrophobic and adopt an α-helical conformation. These stretches are typically around 20 amino acids long and are known as transmembrane (TM) helices. In this review, we will consider how transmembrane helices achieve membrane integration. We will address questions such as: Where do the stretches of amino acids fold into a helical conformation? What is/are the route/routes that these stretches take from synthesis at the ribosome to integration through the ER translocon? How do these stretches 'know' to integrate and in which orientation? How do marginally hydrophobic stretches of amino acids integrate and survive as transmembrane helices?