C-terminal Β-strand Research Articles

The Assembly of the Inverse Autotransporter Protein YeeJ is Driven by its C-terminal β-strand

Autotransporter proteins are bacterial outer membrane proteins that display passenger domains with various functions through a β-barrel shaped translocation domain. YeeJ is an autotransporter protein from E. coli MG1655. In contrast to most other autotransporter proteins, its passenger domain is located at the C-terminus of the translocation domain. Due to this inverted domain organization, YeeJ belongs to autotransporter proteins of type Ve. To investigate the assembly of YeeJ, the fluorescence of a heterologous mCherry passenger domain was measured to quantify its assembly. Based on AlphaFold2 models of 119 sequences similar to YeeJ, a sequence conservation logo for the β1- and the β12-strand of type Ve autotransporter proteins was generated. Then, the effect of mutations in these strands on the assembly of YeeJ were analyzed. Mutations of the N-terminal aromatic amino acid of the β1-strand did not affect the assembly of the translocation domain and the display of the passenger domain. Likewise, exchange of the β1-strand with the β3-strand did not impair the assembly of the autotransporter fusion protein. Mutation of the C-terminal aromatic amino acid of the β12-strand strongly impaired surface display of the mCherry passenger domain. This amino acid has been shown before as an essential feature of the β-signals of classical autotransporter proteins and outer membrane β-barrel proteins in general. We therefore propose that the β12-strand of YeeJ acts as its β-signal and that the assembly of the YeeJ β-barrel is driven by its C-terminal β-strand, like in most other autotransporter proteins, despite its inverted domain organization.

Connecting Protein Millisecond Conformational Dynamics to Protein Thermal Stability.

The stability of protein folded states is crucial for its function, yet the relationship with the protein sequence remains poorly understood. Prior studies have focused on the amino acid composition and thermodynamic couplings within a single folded conformation, overlooking the potential contribution of protein dynamics. Here, we address this gap by systematically analyzing the impact of alanine mutations in the C-terminal β-strand (β5) of ubiquitin, a model protein exhibiting millisecond timescale interconversion between two conformational states differing in the β5 position. Our findings unveil a negative correlation between millisecond dynamics and thermal stability, with alanine substitutions at seemingly flexible C-terminal residues significantly enhancing thermostability. Integrating spectroscopic and computational approaches, we demonstrate that the thermally unfolded state retains a substantial secondary structure but lacks β5 engagement, recapitulating the transition state for millisecond dynamics. Thus, alanine mutations that modulate the stabilities of the folded states with respect to the partially unfolded state impact both the dynamics and stability. Our findings underscore the importance of conformational dynamics with implications for protein engineering and design.

Protein superfolds are characterised as frustration-free topologies: A case study of pure parallel β-sheet topologies.

A protein superfold is a type of protein fold that is observed in at least three distinct, non-homologous protein families. Structural classification studies have revealed a limited number of prevalent superfolds alongside several infrequent occurring folds, and in α/β type superfolds, the C-terminal β-strand tends to favor the edge of the β-sheet, while the N-terminal β-strand is often found in the middle. The reasons behind these observations, whether they are due to evolutionary sampling bias or physical interactions, remain unclear. This article offers a physics-based explanation for these observations, specifically for pure parallel β-sheet topologies. Our investigation is grounded in several established structural rules that are based on physical interactions. We have identified "frustration-free topologies" which are topologies that can satisfy all the rules simultaneously. In contrast, topologies that cannot are termed "frustrated topologies." Our findings reveal that frustration-free topologies represent only a fraction of all theoretically possible patterns, these topologies strongly favor positioning the C-terminal β-strand at the edge of the β-sheet and the N-terminal β-strand in the middle, and there is significant overlap between frustration-free topologies and superfolds. We also used a lattice protein model to thoroughly investigate sequence-structure relationships. Our results show that frustration-free structures are highly designable, while frustrated structures are poorly designable. These findings suggest that superfolds are highly designable due to their lack of frustration, and the preference for positioning C-terminal β-strands at the edge of the β-sheet is a direct result of frustration-free topologies. These insights not only enhance our understanding of sequence-structure relationships but also have significant implications for de novo protein design.

Split Luciferase Molecular Tension Sensors for Bioluminescent Readout of Mechanical Forces in Biological Systems.

The ability of proteins to sense and transmit mechanical forces underlies many biological processes, but characterizing these forces in biological systems remains a challenge. Existing genetically encoded force sensors typically rely on fluorescence or bioluminescence resonance energy transfer (FRET or BRET) to visualize tension. However, these force sensing modules are relatively large, and interpreting measurements requires specialized image analysis and careful control experiments. Here, we report a compact molecular tension sensor that generates a bioluminescent signal in response to tension. This sensor (termed PILATeS) makes use of the split NanoLuc luciferase and consists of the H. sapiens titin I10 domain with the insertion of a 10-15 amino acid tag derived from the C-terminal β-strand of NanoLuc. Mechanical load across PILATeS mediates exposure of this tag to recruit the complementary split NanoLuc fragment, resulting in force-dependent bioluminescence. We demonstrate the ability of PILATeS to report biologically meaningful forces by visualizing forces at the interface between integrins and extracellular matrix substrates. We further use PILATeS as a genetically encoded sensor of tension experienced by the mechanosensing protein vinculin. We anticipate that PILATeS will provide an accessible means of visualizing molecular-scale forces in biological systems.

Identification of potential aggregation hotspots on Aβ42 fibrils blocked by the anti-amyloid chaperone-like BRICHOS domain

Protein misfolding can generate toxic intermediates, which underlies several devastating diseases, such as Alzheimer’s disease (AD). The surface of AD-associated amyloid-β peptide (Aβ) fibrils has been suggested to act as a catalyzer for self-replication and generation of potentially toxic species. Specifically tailored molecular chaperones, such as the BRICHOS protein domain, were shown to bind to amyloid fibrils and break this autocatalytic cycle. Here, we identify a site on the Aβ42 fibril surface, consisting of three C-terminal β-strands and particularly the solvent-exposed β-strand stretching from residues 26–28, which is efficiently sensed by a designed variant of Bri2 BRICHOS. Remarkably, while only a low amount of BRICHOS binds to Aβ42 fibrils, fibril-catalyzed nucleation processes are effectively prevented, suggesting that the identified site acts as a catalytic aggregation hotspot, which can specifically be blocked by BRICHOS. Hence, these findings provide an understanding how toxic nucleation events can be targeted by molecular chaperones.

First 3-D structural evidence of a native-like intertwined dimer in the acylphosphatase family

Acylphosphatase (AcP, EC 3.6.1.7) is a small model protein conformed by a ferredoxin-like fold, profoundly studied to get insights into protein folding and aggregation processes. Numerous studies focused on the aggregation and/or amyloidogenic properties of AcPs suggest the importance of edge-β-strands in the process. In this work, we present the first crystallographic structure of Escherichia coli AcP (EcoAcP), showing notable differences with the only available NMR structure for this enzyme. EcoAcP is crystalised as an intertwined dimer formed by replacing a single C-terminal β-strand between two protomers, suggesting a flexible character of the C-terminal edge of EcoAcP. Despite numerous works where AcP from different sources have been used as a model system for protein aggregation, our domain-swapped EcoAcP structure is the first 3-D structural evidence of native-like aggregated species for any AcP reported to date, providing clues on molecular determinants unleashing aggregation.

Design of Protegrin-1 Analogs with Improved Antibacterial Selectivity.

Protegrin-1 (PG-1) is a cationic β-hairpin pore-forming antimicrobial peptide having a membranolytic mechanism of action. It possesses in vitro a potent antimicrobial activity against a panel of clinically relevant MDR ESKAPE pathogens. However, its extremely high hemolytic activity and cytotoxicity toward mammalian cells prevent the further development of the protegrin-based antibiotic for systemic administration. In this study, we rationally modulated the PG-1 charge and hydrophobicity by substituting selected residues in the central β-sheet region of PG-1 to design its analogs, which retain a high antimicrobial activity but have a reduced toxicity toward mammalian cells. In this work, eight PG-1 analogs with single amino acid substitutions and five analogs with double substitutions were obtained. These analogs were produced as thioredoxin fusions in Escherichia coli. It was shown that a significant reduction in hemolytic activity without any loss of antimicrobial activity could be achieved by a single amino acid substitution, V16R in the C-terminal β-strand, which is responsible for the PG-1 oligomerization. As the result, a selective analog with a ≥30-fold improved therapeutic index was obtained. FTIR spectroscopy analysis of analog, [V16R], revealed that the peptide is unable to form oligomeric structures in a membrane-mimicking environment, in contrast to wild-type PG-1. Analog [V16R] showed a reasonable efficacy in septicemia infection mice model as a systemic antibiotic and could be considered as a promising lead for further drug design.

In Silico Exploration of Alternative Conformational States of VDAC.

VDAC (Voltage-Dependent Anion-selective Channel) is the primary metabolite pore in the mitochondrial outer membrane (OM). Atomic structures of VDAC, consistent with its physiological "open" state, are β-barrels formed by 19 transmembrane (TM) β-strands and an N-terminal segment (NTERM) that folds inside the pore lumen. However, structures are lacking for VDAC's partially "closed" states. To provide clues about possible VDAC conformers, we used the RoseTTAFold neural network to predict structures for human and fungal VDAC sequences modified to mimic removal from the pore wall or lumen of "cryptic" domains, i.e., segments buried in atomic models yet accessible to antibodies in OM-bound VDAC. Predicted in vacuo structures for full-length VDAC sequences are 19-strand β-barrels similar to atomic models, but with weaker H-bonding between TM strands and reduced interactions between NTERM and the pore wall. Excision of combinations of "cryptic" subregions yields β-barrels with smaller diameters, wide gaps between N- and C-terminal β-strands, and in some cases disruption of the β-sheet (associated with strained backbone H-bond registration). Tandem repeats of modified VDAC sequences also were explored, as was domain swapping in monomer constructs. Implications of the results for possible alternative conformational states of VDAC are discussed.

Modeling intermediates of BamA folding an outer membrane protein

BamA, the core component of the β-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 β-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 β-barrel and its POTRA5 domain and either one, two, three, four, five, or six β-hairpins of EspP. The simulation results support an asymmetric-hybrid-barrel model in which the BamA N-terminal β-strand forms stronger interactions with the substrate OMP than the C-terminal β-strand. A consistent “B”-shaped conformation of the final folding intermediate was observed, and the shape of the substrate β-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 β-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.

Thread-by-thread: modelling the intermediate steps of OMP insertion by BamA

Pathogenic Gram-negative bacteria utilize outer membrane proteins (OMPs) for a number of functions such as nutrient transport, signal transduction, and the export of virulence factors. These OMPs are folded and inserted into the outer membrane by the beta-barrel assembly machinery (BAM) complex, the core component of which is the OMP BamA. Previous computational, experimental, and structural studies have revealed that BamA possesses a dynamic lateral gate at the seam between its N- and C-terminal β-strands.

Design for Solubility May Reveal Induction of Amide Hydrogen/Deuterium Exchange by Protein Self-Association

Structural heterogeneity often constrains the characterization of aggregating proteins to indirect or low-resolution methods, obscuring mechanistic details of association. Here, we report progress in understanding the aggregation of Adnectins, engineered binding proteins with an immunoglobulin-like fold. We rationally design Adnectin solubility and measure amide hydrogen/deuterium exchange (HDX) under conditions that permit transient protein self-association. Protein–protein binding commonly slows rates of HDX; in contrast, we find that Adnectin association may induce faster HDX for certain amides, particularly in the C-terminal β-strand. In aggregation-prone proteins, we identify a pattern of very different rates of amide HDX for residues linked by reciprocal hydrogen bonds in the native structure. These results may be explained by local loss of native structure and formation of an inter-protein interface. Amide HDX induced by self-association, detected here by deliberate modulation of propensity for such interactions, may be a general phenomenon with the potential to expose mechanisms of aggregation by diverse proteins.

A C-Terminally Truncated Variant of Neurospora crassa VDAC Assembles Into a Partially Functional Form in the Mitochondrial Outer Membrane and Forms Multimers in vitro.

The voltage-dependent anion-selective channel (VDAC) is a porin in the mitochondrial outer membrane (MOM). Unlike bacterial porins, several mitochondrial β-barrels comprise an odd number of β-strands, as is the case for the 19-β-stranded VDAC. Previously, a variant of a VDAC from Neurospora crassa, VDAC-ΔC, lacking the predicted 19th β-strand, was found to form gated, anion-selective channels in artificial membranes. In vivo, the two C-terminal β-strands (β18 and β19) in VDAC form a β-hairpin necessary for import from the cytoplasm into mitochondria and the β-signal required for assembly in the mitochondrial outer membrane resides in β19. The current study demonstrated that the putative 18-stranded β-barrel formed by VDAC-ΔC can be imported and assembled in the MOM in vivo and can also partially rescue the phenotype associated with the deletion of VDAC from a strain of N. crassa. Furthermore, when expressed and purified from Escherichia coli, VDAC-ΔC can be folded into a β-strand-rich form in decyl-maltoside. Size exclusion chromatography (SEC) alone or combined with multi-angle light scattering (SEC-MALS) and analytical ultracentrifugation revealed that, unlike full-length VDACs, VDAC-ΔC can self-organize into dimers and higher order oligomers in the absence of sterol.

Structural comparisons of host and African swine fever virus dUTPases reveal new clues for inhibitor development

African swine fever, caused by the African swine fever virus (ASFV), is among the most significant swine diseases. There are currently no effective treatments against ASFV. ASFV contains a gene encoding a dUTPase (E165R), which is required for viral replication in swine macrophages, making it an attractive target for inhibitor development. However, the full structural details of the ASFV dUTPase and those of the comparable swine enzyme are not available, limiting further insights. Herein, we determine the crystal structures of ASFV dUTPase and swine dUTPase in both their ligand-free and ligand-bound forms. We observe that the swine enzyme employs a classical dUTPase architecture made up of three-subunit active sites, whereas the ASFV enzyme employs a novel two-subunit active site. We then performed a comparative analysis of all dUTPase structures uploaded in the Protein Data Bank (PDB), which showed classical and non-classical types were mainly determined by the C-terminal β-strand orientation, and the difference was mainly related to the four amino acids behind motif IV. Thus, our study not only explains the reason for the structural diversity of dUTPase but also reveals how to predict dUTPase type, which may have implications for the dUTPase family. Finally, we tested two dUTPase inhibitors developed for the Plasmodium falciparum dUTPase against the swine and ASFV enzymes. One of these compounds inhibited the ASFV dUTPase at low micromolar concentrations (Kd = 15.6 μM) and with some selectivity (∼2x) over swine dUTPase. In conclusion, our study expands our understanding of the dUTPase family and may aid in the development of specific ASFV inhibitors.

Conformational flexibility within the small domain of human serine racemase.

Serine racemase (SR) is a pyridoxal 5'-phosphate (PLP)-containing enzyme that converts L-serine to D-serine, an endogenous co-agonist for the N-methyl-D-aspartate receptor (NMDAR) subtype of glutamate ion channels. SR regulates D-serine levels by the reversible racemization of L-serine to D-serine, as well as the catabolism of serine by α,β-elimination to produce pyruvate. The modulation of SR activity is therefore an attractive therapeutic approach to disorders associated with abnormal glutamatergic signalling since it allows an indirect modulation of NMDAR function. In the present study, a 1.89 Å resolution crystal structure of the human SR holoenzyme (including the PLP cofactor) with four subunits in the asymmetric unit is described. Comparison of this new structure with the crystal structure of human SR with malonate (PDB entry 3l6b) shows an interdomain cleft that is open in the holo structure but which disappears when the inhibitor malonate binds and is enclosed. This is owing to a shift of the small domain (residues 78-155) in human SR similar to that previously described for the rat enzyme. This domain movement is accompanied by changes within the twist of the central four-stranded β-sheet of the small domain, including changes in the φ-ψ angles of all three residues in the C-terminal β-strand (residues 149-151). In the malonate-bound structure, Ser84 (a catalytic residue) points its side chain at the malonate and is preceded by a six-residue β-strand (residues 78-83), but in the holoenzyme the β-strand is only four residues (78-81) and His82 has φ-ψ values in the α-helical region of the Ramachandran plot. These data therefore represent a crystallographic platform that enables the structure-guided design of small-molecule modulators for this important but to date undrugged target.

Bacterial outer membrane proteins assemble via asymmetric interactions with the BamA \u03b2-barrel

The integration of β-barrel proteins into the bacterial outer membrane (OM) is catalysed by the β-barrel assembly machinery (BAM). The central BAM subunit (BamA) itself contains a β-barrel domain that is essential for OM protein biogenesis, but its mechanism of action is unknown. To elucidate its function, here we develop a method to trap a native Escherichia coli β-barrel protein bound stably to BamA at a late stage of assembly in vivo. Using disulfide-bond crosslinking, we find that the first β-strand of a laterally ‘open’ form of the BamA β-barrel forms a rigid interface with the C-terminal β-strand of the substrate. In contrast, the lipid-facing surface of the last two BamA β-strands forms weaker, conformationally heterogeneous interactions with the first β-strand of the substrate that likely represent intermediate assembly states. Based on our results, we propose that BamA promotes the membrane integration of partially folded β-barrels by a ‘swing’ mechanism.

A single residue switch reveals principles of antibody domain integrity

Despite their importance for antibody architecture and design, the principles governing antibody domain stability are still not understood in sufficient detail. Here, to address this question, we chose a domain from the invariant part of IgG, the CH2 domain. We found that compared with other Ig domains, the isolated CH2 domain is a surprisingly unstable monomer, exhibiting a melting temperature of ∼44 °C. We further show that the presence of an additional C-terminal lysine in a CH2 variant substantially increases the melting temperature by ∼14 °C relative to CH2 WT. To explore the molecular mechanism of this effect, we employed biophysical approaches to probe structural features of CH2. The results revealed that Lys101 is key for the formation of three secondary structure elements: the very C-terminal β-strand and two adjacent α-helices. We also noted that a dipole interaction between Lys101 and the nearby α-helix, is important for stabilizing the CH2 architecture by protecting the hydrophobic core. Interestingly, this interaction between the α-helix and C-terminal charged residues is highly conserved in antibody domains, suggesting that it represents a general mechanism for maintaining their integrity. We conclude that the observed interactions involving terminal residues have practical applications for defining domain boundaries in the development of antibody therapeutics and diagnostics.

Out-of-Register Aβ42 Assemblies as Models for Neurotoxic Oligomers and Fibrils.

We propose a variant of the recently found S-shaped Aβ1-42-motif that is characterized by out-of-register C-terminal β-strands. We show that chains with this structure can form not only fibrils that are compatible with the NMR signals but also barrel-shaped oligomers that resemble the ones formed by the much smaller cylindrin peptides. By running long all-atom molecular dynamics simulations at physiological temperatures with an explicit solvent, we study the stability of these constructs and show that they are plausible models for neurotoxic oligomers. After analyzing the transitions between different assemblies, we suggest a mechanism for amyloid formation in Alzheimer's disease.

Solid-State-NMR-Structure-Based Inhibitor Design to Achieve Selective Inhibition of the Parallel-in-Register β-Sheet versus Antiparallel Iowa Mutant β-Amyloid Fibrils.

Solid-state nuclear magnetic resonance (ssNMR) spectroscopy has been widely applied to characterize the high-resolution structures of β-amyloid (Aβ) fibrils. While these structures provide crucial molecular insights on the deposition of amyloid plaques in Alzheimer's diseases (AD), ssNMR structures have been rarely used so far as the basis for designing inhibitors. It remains a challenge because the ssNMR-based Aβ fibril structures were usually obtained with sparsely isotope-labeled peptides with limited experimental constraints, where the structural models, especially the side-chain coordinates, showed restricted precision. However, these structural models often possess a higher accuracy within the hydrophobic core regions with more well-defined experimental data, which provide potential targets for the molecular design. This work presents an ssNMR-based molecular design to achieve selective inhibition of a particular type of Aβ fibrillar structure, which was formed with the Iowa mutant of Aβ with parallel-in-register β-sheet hydrophobic core. The results show that short peptides that mimic the C-terminal β-strands of the fibril may have a preference in binding to the parallel Aβ fibrils rather than the antiparallel fibrils, mainly due to the differences in the high-resolution structures in the fibril elongation interfaces. The Iowa mutant Aβ fibrils are utilized in this work mainly as a model to demonstrate the feasibility of the strategy because it is relatively straightforward to distinguish the parallel and antiparallel fibril structures using ssNMR. Our results suggest that it is potentially feasible to design structure-selective inhibitors and/or diagnostic agents to Aβ fibrils using ssNMR-based structural models.

Sensory domain contraction in histidine kinase CitA triggers transmembrane signaling in the membrane-bound sensor

Bacteria use membrane-integral sensor histidine kinases (HK) to perceive stimuli and transduce signals from the environment to the cytosol. Information on how the signal is transmitted across the membrane by HKs is still scarce. Combining both liquid- and solid-state NMR, we demonstrate that structural rearrangements in the extracytoplasmic, citrate-sensing Per-Arnt-Sim (PAS) domain of HK CitA are identical for the isolated domain in solution and in a longer construct containing the membrane-embedded HK and lacking only the kinase core. We show that upon citrate binding, the PAS domain contracts, resulting in a shortening of the C-terminal β-strand. We demonstrate that this contraction of the PAS domain, which is well characterized for the isolated domain, is the signal transmitted to the transmembrane (TM) helices in a CitA construct in liposomes. Putting the extracytoplasmic PAS domain into context of the membrane-embedded CitA construct slows down citrate-binding kinetics by at least a factor of 60, confirming that TM helix motions are linked to the citrate-binding event. Our results are confirmation of a hallmark of the HK signal transduction mechanism with atomic resolution on a full-length construct lacking only the kinase core domain.

Mechanistic basis for the recognition of a misfolded protein by the molecular chaperone Hsp90.

The critical toxic species in over 40 human diseases are misfolded proteins. Their interaction with molecular chaperones such as Hsp90, which preferentially interacts with metastable proteins, is essential for the blocking of disease progression. Here we used nuclear magnetic resonance (NMR) spectroscopy to determine the three-dimensional structure of the misfolded cytotoxic monomer of the amyloidogenic human protein transthyretin, which is characterized by the release of the C-terminal β-strand and perturbations of the A-B loop. The misfolded transthyretin monomer, but not the wild-type protein, binds to human Hsp90. In the bound state, the Hsp90 dimer predominantly populates an open conformation, and transthyretin retains its globular structure. The interaction surface for the transthyretin monomer comprises the N-terminal and middle domains of Hsp90 and overlaps with that of the Alzheimer's-disease-related protein tau. Taken together, the data suggest that Hsp90 uses a mechanism for the recognition of aggregation-prone proteins that is largely distinct from those of other Hsp90 clients.