C-terminal Interface Research Articles

First-principles study on the interfacial electronic structure and analysis of the bonding properties of NbC/Fe composites

This paper investigated the bonding mode and atoms’ surface energy in the α-Fe and NbC bulk phases in NbC/Fe composites using the first-principles calculation of density functional theory. Different interface models were established to evaluate the interface bonding strength and stability considering the total interface energy and work of adhesion. After structural optimization, four interface models were established, α-Fe(100)/NbC(100) at the C-terminus, α-Fe(100)/NbC(100) at the Nb-terminus, α-Fe(100)/NbC(111) at the C-terminus, and α-Fe(100)/NbC(111) at the Nb-terminus, defined as interfaces I, II, III, and IV, respectively. The results show that the interface adhesion of the C-terminal interface is higher than the Nb-terminal interface. The minimum total energy of interface I and the maximum adhesion work are − 1.80 × 104 eV and 5.31 J/m2, respectively, indicating the most stable interfacial bonding method. The effects of different terminal atoms on the electronic structure and bonding characteristics of the α-Fe(100)/NbC(100) interface were analyzed. The bonding characteristics of the atomic interfaces I and II of the two interface models demonstrated covalent binding of different intensities. Interface I depends on the covalent-ionic bond formed by Fe and C atoms, while interface II relies on metal bonds formed by Fe and Nb atoms and weaker Fe-C bonds. Mulliken's analysis revealed that the bond population of Fe-Nb at interfaces I and II were negative, indicating an intense anti-bonding state between Fe and Nb atoms and a severe anti-bonding effect of Fe-Nb at interface II, resulting in its structural instability. Moreover, because of the covalent bonding (Fe-C) at interface I, the binding strength of the interface was higher with a stable interface.

Structure of dimeric lipoprotein lipase reveals a pore adjacent to the active site

Lipoprotein lipase (LPL) hydrolyzes triglycerides from circulating lipoproteins, releasing free fatty acids. Active LPL is needed to prevent hypertriglyceridemia, which is a risk factor for cardiovascular disease (CVD). Using cryogenic electron microscopy (cryoEM), we determined the structure of an active LPL dimer at 3.9 Å resolution. This structure reveals an open hydrophobic pore adjacent to the active site residues. Using modeling, we demonstrate that this pore can accommodate an acyl chain from a triglyceride. Known LPL mutations that lead to hypertriglyceridemia localize to the end of the pore and cause defective substrate hydrolysis. The pore may provide additional substrate specificity and/or allow unidirectional acyl chain release from LPL. This structure also revises previous models on how LPL dimerizes, revealing a C-terminal to C-terminal interface. We hypothesize that this active C-terminal to C-terminal conformation is adopted by LPL when associated with lipoproteins in capillaries.

Early stage β-amyloid-membrane interactions modulate lipid dynamics and influence structural interfaces and fibrillation

Molecular interactions between β-amyloid (Aβ) peptide and membranes contribute to the neuronal toxicity of Aβ and the pathology of Alzheimer’s disease. Neuronal plasma membranes serve as biologically relevant environments for the Aβ aggregation process as well as affect the structural polymorphisms of Aβ aggregates. However, the nature of these interactions is unknown. Here, we utilized solid-state NMR spectroscopy to explore the site-specific interactions between Aβ peptides and lipids in synaptic plasma membranes at the membrane-associated nucleation stage. The key results show that different segments in the hydrophobic sequence of Aβ initiate membrane binding and interstrand assembling. We demonstrate early stage Aβ-lipid interactions modulate lipid dynamics, leading to more rapid lipid headgroup motion and reduced lateral diffusive motion. These early events influence the structural polymorphisms of yielded membrane-associated Aβ fibrils with distinct C-terminal quaternary interface structure compared to fibrils grown in aqueous solutions. Based on our results, we propose a schematic mechanism by which Aβ-lipid interactions drive membrane-associated nucleation processes, providing molecular insights into the early events of fibrillation in biological environments.

Large protein complex interfaces have evolved to promote cotranslational assembly.

Assembly pathways of protein complexes should be precise and efficient to minimise misfolding and unwanted interactions with other proteins in the cell. One way to achieve this efficiency is by seeding assembly pathways during translation via the cotranslational assembly of subunits. While recent evidence suggests that such cotranslational assembly is widespread, little is known about the properties of protein complexes associated with the phenomenon. Here, using a combination of proteome-specific protein complex structures and publicly available ribosome profiling data, we show that cotranslational assembly is particularly common between subunits that form large intermolecular interfaces. To test whether large interfaces have evolved to promote cotranslational assembly, as opposed to cotranslational assembly being a non-adaptive consequence of large interfaces, we compared the sizes of first and last translated interfaces of heteromeric subunits in bacterial, yeast, and human complexes. When considering all together, we observe the N-terminal interface to be larger than the C-terminal interface 54% of the time, increasing to 64% when we exclude subunits with only small interfaces, which are unlikely to cotranslationally assemble. This strongly suggests that large interfaces have evolved as a means to maximise the chance of successful cotranslational subunit binding.

FCHSD2 cooperates with CDC42 and N-WASP to regulate cell protrusion formation

Actin-based, finger-like cell protrusions such as microvilli and filopodia play important roles in epithelial cells. Several proteins have been identified to regulate cell protrusion formation, which helps us to learn about the underlying mechanism of this process. FCH domain and double SH3 domains containing protein 2 (FCHSD2) belongs to the FCH and Bin-Amphiphysin-Rvs (F-BAR) protein family, containing an N-terminal F-BAR domain, two SH3 domains, and a C-terminal PDZ domain-binding interface (PBI). Previously, we found that FCHSD2 interacts with WASP/N-WASP and stimulates ARP2/3-mediated actin polymerization in vitro. In the present work, we show that FCHSD2 promotes the formation of apical and lateral cell protrusions in cultured cells. Our data suggest that FCHSD2 cooperates with CDC42 and N-WASP in regulating apical cell protrusion formation. In line with this, biochemical studies reveal that FCHSD2 and CDC42 simultaneously bind to N-WASP, forming a protein complex. Interestingly, the F-BAR domain of FCHSD2 induces lateral cell protrusion formation independently of N-WASP. Furthermore, we show that the ability of FCHSD2 to induce cell protrusion formation requires its plasma membrane-binding ability. In summary, our present work suggests that FCHSD2 cooperates with CDC42 and N-WASP to regulate cell protrusion formation in a membrane-dependent manner.

Met35 Oxidation Hinders Aβ25-35 Peptide Aggregation within the Dimyristoylphosphatidylcholine Bilayer.

Using all-atom explicit solvent replica exchange molecular dynamics simulations, we studied the aggregation of oxidized (ox) Aβ25-35 peptides into dimers mediated by the zwitterionic dimyristoylphosphatidylcholine (DMPC) lipid bilayer. By comparing oxAβ25-35 aggregation with that observed for reduced and phosphorylated Aβ25-35 peptides, we elucidated plausible impact of post-translational modifications on cytotoxicity of Aβ peptides involved in Alzheimer's disease. We found that Met35 oxidation reduces helical propensity in oxAβ25-35 peptides bound to the lipid bilayer and enhances backbone fluctuations. These factors destabilize the wild-type head-to-tail dimer interface and lower the aggregation propensity. Met35 oxidation diversifies aggregation pathways by adding monomeric species to the bound conformational ensemble. The oxAβ25-35 dimer becomes partially expelled from the DMPC bilayer and as a result inflicts limited disruption to the bilayer structure compared to wild-type Aβ25-35. Interestingly, the effect of Ser26 phosphorylation is largely opposite, as it preserves the wild-type head-to-tail aggregation interface and strengthens, not weakens, aggregation propensity. The differing effects can be attributed to the sequence locations of these post-translational modifications, since in contrast to Ser26 phosphorylation, Met35 oxidation directly affects the wild-type C-terminal aggregation interface. A comparison with experimental data is provided.

The structure of an Hsp90-immunophilin complex reveals cochaperone recognition of the client maturation state

The Hsp90 chaperone promotes folding and activation of hundreds of client proteins in the cell through an ATP-dependent conformational cycle guided by distinct cochaperone regulators. The FKBP51 immunophilin binds Hsp90 with its tetratricopeptide repeat (TPR) domain and catalyzes peptidyl-prolyl isomerase (PPIase) activity during folding of kinases, nuclear receptors, and tau. Here we determined the cryoelectron microscopy (cryo-EM) structure of the human Hsp90:FKBP51:p23 complex to 3.3Å, which, together with mutagenesis and crosslinking analyses, reveals the basis for cochaperone binding to Hsp90 during client maturation. A helix extension in the TPR functions as a key recognition element, interacting across the Hsp90 C-terminal dimer interface presented in the closed, ATP conformation. The PPIase domain is positioned along the middle domain, adjacent to Hsp90 client binding sites, whereas a single p23 makes stabilizing interactions with the N-terminal dimer. With this architecture, FKBP51 is positioned to act on specific client residues presented during Hsp90-catalyzed remodeling.

RyR2 disease mutations at the C-terminal domain intersubunit interface alter closed-state stability and channel activation

Ryanodine receptors (RyRs) are ion channels that mediate the release of Ca2+ from the sarcoplasmic reticulum/endoplasmic reticulum, mutations of which are implicated in a number of human diseases. The adjacent C-terminal domains (CTDs) of cardiac RyR (RyR2) interact with each other to form a ring-like tetrameric structure with the intersubunit interface undergoing dynamic changes during channel gating. This mobile CTD intersubunit interface harbors many disease-associated mutations. However, the mechanisms of action of these mutations and the role of CTD in channel function are not well understood. Here, we assessed the impact of CTD disease-associated mutations P4902S, P4902L, E4950K, and G4955E on Ca2+− and caffeine-mediated activation of RyR2. The G4955E mutation dramatically increased both the Ca2+-independent basal activity and Ca2+-dependent activation of [3H]ryanodine binding to RyR2. The P4902S and E4950K mutations also increased Ca2+ activation but had no effect on the basal activity of RyR2. All four disease mutations increased caffeine-mediated activation of RyR2 and reduced the threshold for activation and termination of spontaneous Ca2+ release. G4955D dramatically increased the basal activity of RyR2, whereas G4955K mutation markedly suppressed channel activity. Similarly, substitution of P4902 with a negatively charged residue (P4902D), but not a positively charged residue (P4902K), also dramatically increased the basal activity of RyR2. These data suggest that electrostatic interactions are involved in stabilizing the CTD intersubunit interface and that the G4955E disease mutation disrupts this interface, and thus the stability of the closed state. Our studies shed new insights into the mechanisms of action of RyR2 CTD disease mutations.

Molecular Switch between Structural Compaction and Thermodynamic Stability by the Xxx-Pro Interface in Transmembrane β-Barrels.

Transmembrane β-barrel scaffolds found in outer membrane proteins are formed and stabilized by a defined pattern of interstrand intraprotein H-bonds, in hydrophobic lipid bilayers. Introducing the conformationally constrained proline in β-barrels can cause significant destabilization of these structural regions that require H-bonding, with proline additionally acting as a secondary structure breaker. Membrane protein β-barrels are therefore expected to show poor tolerance to the presence of a transmembrane proline. Here, we assign a thermodynamic measure for the extent to which a single proline can be tolerated at the C-terminal interface of the model transmembrane β-barrel PagP. We find that proline drastically destabilizes PagP by 7.0 kcal mol-1 with respect to wild-type PagP (F161 → P161). Interestingly, strategic modulation of the preceding residue can modify the measured energetics. Placing a hydrophobic or bulky side chain as the preceding residue increases the thermodynamic stability by ≤8.0 kcal mol-1. While polar substituents at the preceding residue decrease the PagP stability, these residues demonstrate stronger tertiary packing interactions in the barrel and retain the catalytic activity of native PagP. This biophysical interplay between enhanced thermodynamic stability and attaining a structurally compact functional β-barrel scaffold highlights the detrimental effect caused by proline incorporation. Our findings also reveal alternative mechanisms that protein sequences can employ to salvage the structural integrity of transmembrane protein structures.

Structure based virtual screening of natural products to disrupt the structural integrity of TRAF6 C-terminal domain homotrimer

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an E3 ligase which takes part in different cellular pathways. TRAF6 is seen to be highly expressed in various cancers and most importantly is known to drive cancer metastasis. This makes TRAF6 a potential therapeutic target. In our previous studies, we observed that the C-terminal domain of TRAF6 forms a mushroom shaped trimer structure. Lys340 and Glu345 were identified to be the most critical residues in the trimer interface. In this current work, we screened for more than 14000 small molecules derived from various natural sources and they were screened against TRAF6 C-terminal trimer interaction interface to prevent the formation of the interface. All the obtained molecules were tested for their drug-likeliness properties. The ligands which qualified the filter were considered for protein-ligand docking or structure based virtual screening in GOLD 5.2. Pose selection was carried out on the basis of GoldScore and ChemScore function of GOLD 5.2. Top 20 molecules binding the TRAF6 trimeric interface were tested for their ADME properties. From the top 20 molecules, top 3 ligands were chosen based on their abilities to pass the maximum numbers of ADME filters.

The heme-sensitive regulator SbnI has a bifunctional role in staphyloferrin B production by Staphylococcus aureus

Staphylococcus aureus infection relies on iron acquisition from its host. S. aureus takes up iron through heme uptake by the iron-responsive surface determinant (Isd) system and by the production of iron-scavenging siderophores. Staphyloferrin B (SB) is a siderophore produced by the 9-gene sbn gene cluster for SB biosynthesis and efflux. Recently, the ninth gene product, SbnI, was determined to be a free l-serine kinase that produces O-phospho-l-serine (OPS), a substrate for SB biosynthesis. Previous studies have also characterized SbnI as a DNA-binding regulatory protein that senses heme to control sbn gene expression for SB synthesis. Here, we present crystal structures at 1.9-2.1 Å resolution of a SbnI homolog from Staphylococcus pseudintermedius (SpSbnI) in both apo form and in complex with ADP, a product of the kinase reaction; the latter confirmed the active-site location. The structures revealed that SpSbnI forms a dimer through C-terminal domain swapping and a dimer of dimers through intermolecular disulfide formation. Heme binding had only a modest effect on SbnI enzymatic activity, suggesting that its two functions are independent and structurally distinct. We identified a heme-binding site and observed catalytic heme transfer between a heme-degrading protein of the Isd system, IsdI, and SbnI. These findings support the notion that SbnI has a bifunctional role contributing precursor OPS to SB synthesis and directly sensing heme to control expression of the sbn locus. We propose that heme transfer from IsdI to SbnI enables S. aureus to control iron source preference according to the sources available in the environment.

Role of Diffusion in Unfolding and Translocation of Multidomain Titin I27 Substrates by a Clp ATPase Nanomachine.

Degradation of multidomain substrate proteins (SPs) by AAA+ nanomachines takes place processively from the tagged terminal. Ring-shaped ATPase components, such as ClpY, apply repetitive mechanical forces to effect domain unfolding and translocation of polypeptide segments through a narrow central channel. We study these mechanisms through atomistic Langevin dynamics simulations of C-terminal-tagged SPs in allosteric ClpY cycles. We find that monomeric SPs are processed through single unfolding pathways and fast timescales, whereas multimeric SPs involve branched pathways and slower timescales. These distinct mechanisms are attributed to the slower rotational diffusion of the C-terminal domain in multidomain SPs that hinders access to the soft mechanical direction. In the geometry specific to laser optical tweezers experiments, involving a restrained SP N-terminal, a single unfolding pathway is found for both monomeric and tetrameric SPs as pulling is applied along the N-C direction. Non-native interactions modulate unfolding of unrestrained monomers by weakening the C-terminal interface but do not contribute significantly to unfolding of restrained SPs. On the basis of these results, we propose that the interplay of restricted SP dynamics and ATPase kinetics underlies partitioning of multidomain SPs into completely degraded products and undegraded fragments comprising folded domains.

Salvaging the Thermodynamic Destabilization of Interface Histidine in Transmembrane β-Barrels.

The ability of histidine to participate in a wide range of stabilizing polar interactions preferentially populates this residue in functionally important sites of proteins. Histidine possesses an amphiphilic and electrostatic nature that is essential for amino acids residing at membrane interfaces. However, the frequency of occurrence of histidine at membrane interfaces, particularly transmembrane β-barrels, is lower than those of other aromatic residues. Here, we carry out comprehensive energetic measurements using equilibrium folding of the outer membrane enzyme PagP to address the contribution of a C-terminal interface histidine to barrel stability. We show that placing histidine at the C-terminus universally destabilizes PagP by 4.0–8.0 kcal mol–1 irrespective of the neighboring residue. Spectroscopic and electrophoretic measurements indicate that the altered stability may arise from a loss of barrel compaction. Isoleucine, methionine, and valine salvage this destabilization marginally (in addition to tyrosine, which shows an exceptionally high folding free energy value), when placed at the penultimate position, at the expense of an altered folding pathway. Double-mutant cycle analysis indicates that the coupling energy between the terminal and penultimate residues in PagP-X160H161 increases when the level of intrinsic destabilization by the terminal H161 is high. Our observations that neighboring residues cannot salvage the energetic destabilization of histidine may explain why histidine is less abundant at membrane interfaces.

Nucleotide-Dependent Dimer Association and Dissociation of the Chaperone Hsp90.

Hsp90 is an essential molecular chaperone, which has to be in a dimeric form for its correct function. While the affinity of the dimer has previously been measured, little is known about how it associates and dissociates and the factors that influence this. We perform an in-depth single molecule characterization of the C-terminal association and dissociation of Hsp90. We find more than one dissociation rate, indicating that the dimer has a stable and an unstable state. Furthermore, we find that the stability of the C-terminal association is dependent on the presence of ATP, despite the C-terminal dimerization interface being distal to the catalytic site.

Cotranslational protein assembly imposes evolutionary constraints on homomeric proteins.

Cotranslational protein folding can facilitate rapid formation of functional structures. However, it can also cause premature assembly of protein complexes, if two interacting nascent chains are in close proximity. By analyzing known protein structures, we show that homomeric protein contacts are enriched toward the C termini of polypeptide chains across diverse proteomes. We hypothesize that this is the result of evolutionary constraints for folding to occur before assembly. Using high-throughput imaging of protein homomers in Escherichia coli and engineered protein constructs with N- and C-terminal oligomerization domains, we show that, indeed, proteins with C-terminal homomeric interface residues consistently assemble more efficiently than those with N-terminal interface residues. Using in vivo, in vitro and in silico experiments, we identify features that govern successful assembly of homomers, which have implications for protein design and expression optimization.

Structural Insights into Yeast Telomerase Recruitment to Telomeres

Telomerase maintains chromosome ends from humans to yeasts. Recruitment of yeast telomerase to telomeres occurs through its Ku and Est1 subunitsvia independent interactions with telomeraseRNA(TLC1) and telomeric proteins Sir4 and Cdc13,respectively. However, the structures of the molecules comprising these telomerase-recruiting pathways remain unknown. Here, we report crystal structures of the Ku heterodimer and Est1 complexed with their key binding partners. Two major findings are as follows: (1) Ku specifically binds to telomerase RNA in a distinct, yet related, manner to how it binds DNA; and (2) Est1 employs two separate pockets to bind distinct motifs of Cdc13. The N-terminal Cdc13-binding site of Est1 cooperates with the TLC1-Ku-Sir4 pathway for telomerase recruitment, whereas the C-terminal interface is dispensable for binding Est1 invitro yet is nevertheless essential for telomere maintenance invivo. Overall, our results integrate previous models and provide fundamentally valuable structural information regarding telomere biology.

A Diverse Set of Single-domain Antibodies (VHHs) against the Anthrax Toxin Lethal and Edema Factors Provides a Basis for Construction of a Bispecific Agent That Protects against Anthrax Infection

Infection with Bacillus anthracis, the causative agent of anthrax, can lead to persistence of lethal secreted toxins in the bloodstream, even after antibiotic treatment. VHH single-domain antibodies have been demonstrated to neutralize diverse bacterial toxins both in vitro and in vivo, with protein properties such as small size and high stability that make them attractive therapeutic candidates. Recently, we reported on VHHs with in vivo activity against the protective antigen component of the anthrax toxins. Here, we characterized a new set of 15 VHHs against the anthrax toxins that act by binding to the edema factor (EF) and/or lethal factor (LF) components. Six of these VHHs are cross-reactive against both EF and LF and recognize the N-terminal domain (LFN, EFN) of their target(s) with subnanomolar affinity. The cross-reactive VHHs block binding of EF/LF to the protective antigen C-terminal binding interface, preventing toxin entry into the cell. Another VHH appears to recognize the LF C-terminal domain and exhibits a kinetic effect on substrate cleavage by LF. A subset of the VHHs neutralized against EF and/or LF in murine macrophage assays, and the neutralizing VHHs that were tested improved survival of mice in a spore model of anthrax infection. Finally, a bispecific VNA (VHH-based neutralizing agent) consisting of two linked toxin-neutralizing VHHs, JMN-D10 and JMO-G1, was fully protective against lethal anthrax spore infection in mice as a single dose. This set of VHHs should facilitate development of new therapeutic VNAs and/or diagnostic agents for anthrax.

Plasma Membrane Targeting of Protocadherin 15 Is Regulated by the Golgi-Associated Chaperone Protein PIST.

Protocadherin 15 (PCDH15) is a core component of hair cell tip-links and crucial for proper function of inner ear hair cells. Mutations of PCDH15 gene cause syndromic and nonsyndromic hearing loss. At present, the regulatory mechanisms responsible for the intracellular transportation of PCDH15 largely remain unknown. Here we show that PIST, a Golgi-associated, PDZ domain-containing protein, interacts with PCDH15. The interaction is mediated by the PDZ domain of PIST and the C-terminal PDZ domain-binding interface (PBI) of PCDH15. Through this interaction, PIST retains PCDH15 in the trans-Golgi network (TGN) and reduces the membrane expression of PCDH15. We have previously showed that PIST regulates the membrane expression of another tip-link component, cadherin 23 (CDH23). Taken together, our finding suggests that PIST regulates the intracellular trafficking and membrane targeting of the tip-link proteins CDH23 and PCDH15.

Structural Characterization of Bardet-Biedl Syndrome 9 Protein (BBS9)

The Bardet-Biedl syndrome protein complex (BBSome) is an octameric complex that transports membrane proteins into the primary cilium signaling organelle in eukaryotes and is implicated in human disease. Here we have analyzed the 99-kDa human BBS9 protein, one of the eight BBSome components. The protein is composed of four structured domains, including a β-stranded N-terminal domain. The 1.8 Å crystal structure of the 46-kDa N-terminal domain reveals a seven-bladed β-propeller. A structure-based homology search suggests that it functions in protein-protein interactions. We show that the Bardet-Biedl syndrome-causing G141R mutation in BBS9 likely results in misfolding of the β-propeller. Although the C-terminal half of BBS9 dimerizes in solution, the N-terminal domain only does so in the crystal lattice. This C-terminal dimerization interface might be important for the assembly of the BBSome.

High-resolution Crystal Structure of Dimeric VP40 From Sudan ebolavirus.

Ebolaviruses cause severe hemorrhagic fever. Central to the Ebola life cycle is the matrix protein VP40, which oligomerizes and drives viral budding. Here we present the crystal structure of the Sudan virus (SUDV) matrix protein. This structure is higher resolution (1.6 Å) than previously achievable. Despite differences in the protein purification, we find that it still forms a stable dimer in solution, as was noted for other Ebola VP40s. Although the N-terminal domain interface by which VP40 dimerizes is conserved between Ebola virus and SUDV, the C-terminal domain interface by which VP40 dimers may further assemble is significantly smaller in this SUDV assembly.