Search Results (116)

Cold environments challenge the structural and functional integrity of membrane proteins, requiring specialized adaptations to maintain activity under low thermal energy. Here, we investigate the molecular basis of cold tolerance in the peptide transporter PepT1 from the Antarctic icefish (Chionodraco hamatus, ChPepT1) using molecular dynamics simulations, binding free energy calculations (MM/GBSA), and dynamic network analysis. We compare ChPepT1 to its human ortholog (hPepT1), a non-cold-adapted variant, to reveal key features enabling psychrophilic function. Our simulations show that ChPepT1 displays enhanced global flexibility, particularly in domains adjacent to the substrate-binding site and the C-terminal domain (CTD). While hPepT1 loses substrate binding affinity as temperature increases, ChPepT1 maintains stable peptide interactions across a broad thermal range. This thermodynamic buffering results from temperature-sensitive rearrangement of hydrogen bond networks and more dynamic lipid interactions. Importantly, we identify a temperature-responsive segment (TRS, residues 660–670) within the proximal CTD that undergoes an α-helix to coil transition, modulating long-range coupling with transmembrane helices. Dynamic cross-correlation analyses further suggest that ChPepT1, unlike hPepT1, reorganizes its interdomain communication in response to temperature shifts. Our findings suggest that cold tolerance in ChPepT1 arises from a combination of structural flexibility, resilient substrate binding, and temperature-sensitive interdomain dynamics. These results provide new mechanistic insight into thermal adaptation in membrane transporters and offer a framework for engineering proteins with enhanced functionality in extreme environments. Full article

The sodium/proton exchanger NHA2, also known as SLC9B2, is important for insulin secretion, renal blood pressure regulation, and electrolyte retention. Recent structures of bison NHA2 has revealed its unique 14-transmembrane helix architecture, which is different from SLC9A/NHE members made up from 13-TM helices. Sodium/proton exchangers are functional homodimers, and the additional N-terminal helix in NHA2 was found to alter homodimer assembly. Here, we present the cryo-electron microscopy structures of apo human NHA2 in complex with a Fab fragment and also with the inhibitor phloretin bound at 2.8 and 2.9 Å resolution, respectively. We show how phosphatidic acid (PA) lipids bind to the homodimer interface of NHA2 on the extracellular side, which we propose has a regulatory role linked to cell volume regulation. The ion binding site of human NHA2 has a salt bridge interaction between the ion binding aspartate D278 and R432, an interaction previously broken in the bison NHA2 structure, and these differences suggest a possible ion coupling mechanism. Lastly, the human NHA2 structure in complex with phloretin offers a template for structure-guided drug design, potentially leading to the development of more selective and potent NHA2 inhibitors. Full article

Microorganisms exert antagonistic effects on pathogens through different mechanisms, thereby achieving biological control of plant diseases. Many Burkholderia strains can produce complex secondary metabolites and substances that have toxic effects on host cells. The phage tail-like bacteriocins (tailocins) is a compound with antibacterial activity. However, its function in B. multivorans has not yet been reported. This article explores the ability of B. multivorans WS-FJ9 to antagonise plant pathogenic fungi and oomycetes, screening the potential tailocins in the strain WS-FJ9 and verifying their function, to reveal its novel antimicrobial mechanisms. We found that WS-FJ9 had strong antagonistic effects on the plant pathogenic fungi Phomopsis macrospore and Sphaeropsis sapinea, and the pathogenic oomycete Phytophthora cinnamomi. The phage tail-like protein Bm_67459 was predicted from the WS-FJ9 strain genome. The Bm_67459 cDNA encoded 111 amino acid sequence, and the relative molecular weight was approximately 11.69 kDa, the theoretical isoelectric point (pI) was 5.49, and it was a hydrophilic protein. Bm_67459 had no transmembrane helix region or signal peptide, and it belonged to the Phage_TAC_7 super family. qRT-PCR results showed that Bm_67459 gene expression was significantly upregulated during contact between WS-FJ9 and P. cinnamomi. The purified Bm_67459 protein significantly inhibited P. cinnamomi mycelial growth at 10 μg·mL⁻¹. In summary, the WS-FJ9 strain had broad-spectrum anti-phytopathogenic activity, and the tailocin Bm_67459 was an important effector against the plant pathogen P. cinnamomi, which helps to reveal the antagonistic mechanism of this strain at the molecular level and provides excellent strain resources for the biological control of plant diseases. Full article

G protein-coupled receptors (GPCRs) constitute the largest and most frequently used family of molecular drug targets. The simplicity of GPCR drug design results from their common seven-transmembrane-helix topology and well-understood signaling pathways. GPCRs are extremely sensitive to slight changes in the chemical structure of compounds, which allows for the reliable design of highly selective and specific drugs. Only recently has the number of GPCR structures, both in their active and inactive conformations, together with their active ligands, become sufficient to comprehensively apply machine learning in decision support systems to predict compound activity in drug design. Here, we describe GPCRVS, an efficient machine learning system for the online assessment of the compound activity against several GPCR targets, including peptide- and protein-binding GPCRs, which are the most difficult for virtual screening tasks. As a decision support system, GPCRVS evaluates compounds in terms of their activity range, the pharmacological effect they exert on the receptor, and the binding mode they could demonstrate for different types and subtypes of GPCRs. GPCRVS allows for the evaluation of compounds ranging from small molecules to short peptides provided in common chemical file formats. The results of the activity class assignment and the binding affinity prediction are provided in comparison with predictions for known active ligands of each included GPCR. Multiclass classification in GPCRVS, handling incomplete and fuzzy biological data, was validated on ChEMBL and Google Patents-retrieved data sets for class B GPCRs and chemokine CC and CXC receptors. Full article

Purple acid phosphatases (PAPs) play a key role in phosphorus (P) assimilation and redistribution in plants, catalyzing the hydrolysis of phosphate esters to produce inorganic phosphate (Pi). In this study, a total of 77 PAP genes were identified in B. napus. The candidate genes were divided into three groups and ten subgroups based on the phylogenetic analyses and exon-intron organization. Among these 77 BnaPAP proteins, 35 exhibit typical metal-ligating residues characteristic of known PAPs, whereas certain unaltered amino acid residues were absent or displaced in other BnaPAPs. A computational prediction was conducted, revealing that the majority of PAPs contain signal peptide motifs and display a range of N-glycosylation levels, as well as transmembrane helix motifs. An analysis of previously obtained RNA-seq data revealed that 55.84% (43 of 77) of the BnaPAPs responded to Pi deficiency. Moreover, we conducted a preliminary examination of the expression profiles of BnaPAP genes in response to salt stress, and discovered that 42.86% (33 of 77) of these genes were induced under salt stress, either in the shoots or in the roots. Further qRT-PCR and GUS analyses revealed that BnaC9.PAP10 and BnaA7.PAP10, two paralogs of BnaPAP10s, were induced by Pi deficiency. Notably, BnaC9.PAP10 exhibits robust induction, compared to the relatively mild induction observed in BnaA7.PAP10. Our research shows that BnaA7.PAP10 uniquely responds to Pi stress via the W-box, while BnaA7.PAP10 predominantly responds via the P1BS element, and the differences in cis-regulatory elements (CREs) within their promoter regions specifically contribute to their distinct expression levels under Pi stress. Our findings provide valuable insights and establish a foundation for future functional studies of BnaPAPs. Full article

Alzheimer’s disease (AD) pathogenesis is correlated with the membrane content of various lipid species, including cholesterol, whose interactions with amyloid precursor protein (APP) have been extensively explored. Amyloid-β peptides triggering AD are products of APP cleavage by secretases, which differ depending on the APP and secretase location relative to ordered or disordered membrane microdomains. We used high-resolution NMR to probe the interactions of the cholesterol analog with APP transmembrane domain in two membrane-mimicking systems resembling ordered or perturbed lipid environments (bicelles/micelles). In bicelles, spin-labeled sterol interacted with the peptide near the amphiphilic juxtamembrane region and N-terminal part of APP transmembrane helix, as described earlier for cholesterol. Upon transition into micellar environment, another interaction site appeared where sterol polar head was buried in the hydrophobic core near the hinge region. In MD simulations, sterol moved between three interaction sites, sliding along the polar groove formed by glycine residues composing the dimerization interfaces and flexible hinge of the APP transmembrane domain. Because the lipid environment modulates interactions, the role of lipids in the AD pathogenesis is defined by the state of the entire lipid subsystem rather than the effects of individual lipid species. Cholesterol can interplay with other lipids (polyunsaturated, gangliosides, etc.), determining the outcome of amyloid-β production cascades. Full article

The angiotensin-converting enzyme-2 (ACE2) is a transmembrane glycoprotein, consisting of two segments: a large carboxypeptidase catalytic domain and a small transmembrane collectrin-like segment. This protein plays an essential role in blood pressure regulation, transforming the peptides angiotensin-I and angiotensin-II (vasoconstrictors) into angiotensin-1-9 and angiotensin-1-7 (vasodilators). During the COVID-19 pandemic, ACE2 became best known as the receptor of the S-protein of SARS-CoV-2 coronavirus. The purpose of the following research is to reconstruct the 3D structure of the catalytic domain of the rabbit enzyme rACE2 using its primary amino acid sequence, and then to compare it with the human analog hACE2. For this purpose, we have calculated the electric properties and thermodynamic stability of the two protein globules employing computer programs for protein electrostatics. The analysis of the amino acid content and sequence demonstrates an 85% identity between the two polypeptide chains. The 3D alignment of the catalytic domains of the two enzymes shows coincidence of the α-helix segments, and a small difference in two unstructured segments of the chain. The electric charge of the catalytic domain of rACE2, determined by 70 positively chargeable amino acid residues, 114 negatively chargeable ones, and two positive charges of the Zn²⁺ atom in the active center exceeds that of hACE2 by one positively and four negatively chargeable groups; however, in 3D conformation, their isoelectric points pI 5.21 coincide. The surface electrostatic potential is similarly distributed on the surface of the two catalytic globules, but it strongly depends on the pH of the extracellular medium: it is almost positive at pH 5.0 but strongly negative at pH 7.4. The pH dependence of the electrostatic component of the free energy discloses that the 3D structure of the two enzymes is maximally stable at pH 6.5. The high similarity in the 3D structure, as well as in the electrostatic and thermodynamic properties, suggests that rabbit can be successfully used as an animal model to study blood pressure regulation and coronavirus infection, and the results can be extrapolated to humans. Full article

In this manuscript, a novel presenilin-2 (PSEN2) mutation, Val226Ala, was found in a 59-year-old Korean patient who exhibited rapid progressive memory dysfunction and hallucinations six months prior to her first visit to the hospital. Her Magnetic Resonance Imaging (MRI) showed brain atrophy, and both amyloid positron emission tomography (PET) and multimer detection system-oligomeric amyloid-beta (Aβ) results were positive. The patient was diagnosed with early onset Alzheimer’s disease. The whole-exome analysis revealed a new PSEN2 Val226Ala mutation with heterozygosity in the 5th transmembrane domain of the PSEN2 protein near the lumen region. Analyses of the structural prediction suggested structural changes in the helix, specifically a loss of a hydrogen bond between Val226 and Gln229, which may lead to elevated helix motion. Multiple PSEN2 mutations were reported in PSEN2 transmembrane-5 (TM5), such as Tyr231Cys, Ile235Phe, Ala237Val, Leu238Phe, Leu238Pro, and Met239Thr, highlighting the dynamic importance of the 5th transmembrane domain of PSEN2. Mutations in TM5 may alter the access tunnel of the Aβ substrate in the membrane to the gamma-secretase active site, indicating a possible influence on enzyme function that increases Aβ production. Interestingly, the current patient with the Val226Ala mutation presented with a combination of hallucinations and memory dysfunction. Although the causal mechanisms of hallucinations in AD remain unclear, it is possible that PSEN2 interacts with other disease risk factors, including Notch Receptor 3 (NOTCH3) or Glucosylceramidase Beta-1 (GBA) variants, enhancing the occurrence of hallucinations. In conclusion, the direct or indirect role of PSEN2 Val226Ala in AD onset cannot be ruled out. Full article

An H-bond involves the sharing of a hydrogen atom between an electronegative atom to which it is covalently bound (the donor) and another electronegative atom serving as an acceptor. Such bonds represent a critically important geometrical force in biological macromolecules and, as such, have been characterized extensively. H-bond formation invariably leads to a weakening within the acceptor moiety due to the pulling exerted by the donor hydrogen. This phenomenon can be compared to a spring connecting two masses; pulling one mass stretches the spring, similarly affecting the bond between the two masses. Herein, we describe the opposite phenomenon when investigating the energetics of the C–H···O=C bond. This bond underpins the most prevalent protein transmembrane dimerization motif (GxxxG) in which a glycine Cα-H on one helix forms a hydrogen bond with a carbonyl in a nearby helix. We use isotope-edited FT-IR spectroscopy and corroborating computational approaches to demonstrate a surprising strengthening of the acceptor C=O bond upon binding with the glycine Cα-H. We show that electronic factors associated with the Cα-H bond strengthen the C=O oscillator by increasing the s-character of the σ-bond, lowering the hyperconjugative disruption of the π-bond. In addition, a reduction of the acceptor C=O bond’s polarity is observed upon the formation of the C–H···O=C bond. Our findings challenge the conventional understanding of H-bond dynamics and provide new insights into the structural stability of inter-helical protein interactions. Full article

Residue contact maps provide a condensed two-dimensional representation of three-dimensional protein structures, serving as a foundational framework in structural modeling but also as an effective tool in their own right in identifying inter-helical binding sites and drawing insights about protein function. Treating contact maps primarily as an intermediate step for 3D structure prediction, contact prediction methods have limited themselves exclusively to sequential features. Now that AlphaFold2 predicts 3D structures with good accuracy in general, we examine (1) how well predicted 3D structures can be directly used for deciding residue contacts, and (2) whether features from 3D structures can be leveraged to further improve residue contact prediction. With a well-known benchmark dataset, we tested predicting inter-helical residue contact based on AlphaFold2’s predicted structures, which gave an 83% average precision, already outperforming a sequential features-based state-of-the-art model. We then developed a procedure to extract features from atomic structure in the neighborhood of a residue pair, hypothesizing that these features will be useful in determining if the residue pair is in contact, provided the structure is decently accurate, such as predicted by AlphaFold2. Training on features generated from experimentally determined structures, we leveraged knowledge from known structures to significantly improve residue contact prediction, when testing using the same set of features but derived using AlphaFold2 structures. Our results demonstrate a remarkable improvement over AlphaFold2, achieving over 91.9% average precision for a held-out subset and over 89.5% average precision in cross-validation experiments. Full article

Connexins (Cxs) are a family of integral membrane proteins, which function as both hexameric hemichannels (HCs) and dodecameric gap junction channels (GJCs), behaving as conduits for the electrical and molecular communication between cells and between cells and the extracellular environment, respectively. Their proper functioning is crucial for many processes, including development, physiology, and response to disease and trauma. Abnormal GJC and HC communication can lead to numerous pathological states including inflammation, skin diseases, deafness, nervous system disorders, and cardiac arrhythmias. Over the last 15 years, high-resolution X-ray and electron cryomicroscopy (cryoEM) structures for seven Cx isoforms have revealed conservation in the four-helix transmembrane (TM) bundle of each subunit; an αβ fold in the disulfide-bonded extracellular loops and inter-subunit hydrogen bonding across the extracellular gap that mediates end-to-end docking to form a tight seal between hexamers in the GJC. Tissue injury is associated with cellular Ca²⁺ overload. Surprisingly, the binding of 12 Ca²⁺ ions in the Cx26 GJC results in a novel electrostatic gating mechanism that blocks cation permeation. In contrast, acidic pH during tissue injury elicits association of the N-terminal (NT) domains that sterically blocks the pore in a “ball-and-chain” fashion. The NT domains under physiologic conditions display multiple conformational states, stabilized by protein–protein and protein–lipid interactions, which may relate to gating mechanisms. The cryoEM maps also revealed putative lipid densities within the pore, intercalated among transmembrane α-helices and between protomers, the functions of which are unknown. For the future, time-resolved cryoEM of isolated Cx channels as well as cryotomography of GJCs and HCs in cells and tissues will yield a deeper insight into the mechanisms for channel regulation. The cytoplasmic loop (CL) and C-terminal (CT) domains are divergent in sequence and length, are likely involved in channel regulation, but are not visualized in the high-resolution X-ray and cryoEM maps presumably due to conformational flexibility. We expect that the integrated use of synergistic physicochemical, spectroscopic, biophysical, and computational methods will reveal conformational dynamics relevant to functional states. We anticipate that such a wealth of results under different pathologic conditions will accelerate drug discovery related to Cx channel modulation. Full article

The linear undecapeptide KKLFKKILKYL-NH₂ (BP100) highlights for its antibacterial activity against Gram-negative bacteria and its low toxicity. These excellent biological properties prompted the investigation of its mechanism of action, which were undertaken using spectroscopic techniques, biophysical analysis, microscopy, and molecular dynamic simulations. Studies were conducted in different membrane environments, such as anionic, zwitterionic, and mixed membranes, as well as in vesicles (LUVs and GUVs) and bacteria. The findings suggest that BP100 exhibits a preference for anionic membranes, and its mechanism of action involves charge neutralization and membrane permeabilization. In these membranes, BP100 transitions from an unstructured state in water to an α-helix with the axis parallel to the surface. MD simulations suggest that after electrostatic interaction with the membrane, BP100 flips, facilitating the insertion of its hydrophobic face into the membrane bilayer. Thus, BP100 adopts an almost vertical transmembrane orientation with lysine side chains snorkelling on both sides of the membrane. As a result of the rotation, BP100 induces membrane thinning and slow lipid diffusion and promotes water penetration, particularly in anionic lipid membranes. These investigations pointed towards a carpet-like mechanism and are aligned with the biological activity profile described for BP100. This review covers all the studies carried out on the mechanism of action of BP100 published between 2009 and 2023. Full article

Two novel members of the subfamily Betarhabdovirinae, family Rhabdoviridae, were identified in Brazil. Overall, their genomes have the typical organization 3′-N-P-P3-M-G-L-5′ observed in mono-segmented plant-infecting rhabdoviruses. In aristolochia-associated cytorhabdovirus (AaCV), found in the liana aristolochia (Aristolochia gibertii Hook), an additional short orphan ORF encoding a transmembrane helix was detected between P3 and M. The AaCV genome and inferred encoded proteins share the highest identity values, consistently < 60%, with their counterparts of the yerba mate chlorosis-associated virus (Cytorhabdovirus flaviyerbamate). The second virus, false jalap virus (FaJV), was detected in the herbaceous plant false jalap (Mirabilis jalapa L.) and represents together with tomato betanucleorhabdovirus 2, originally found in tomato plants in Slovenia, a tentative new species of the genus Betanucleorhabdovirus. FaJV particles accumulate in the perinuclear space, and electron-lucent viroplasms were observed in the nuclei of the infected cells. Notably, distinct from typical rhabdoviruses, most virions of AaCV were observed to be non-enclosed within membrane-bounded cavities. Instead, they were frequently seen in close association with surfaces of mitochondria or peroxisomes. Unlike FaJV, AaCV was successfully graft-transmitted to healthy plants of three species of the genus Aristolochia, while mechanical and seed transmission proved unsuccessful for both viruses. Data suggest that these viruses belong to two new tentative species within the subfamily Betarhabdovirinae. Full article

Cannabinoid receptors (CB1 and CB2) are promising targets for a better understanding of neurological diseases. Nevertheless, only a few ligands of CB have reached clinical application so far. Venoms are considered as interesting sources of novel biologically active compounds. Here, we describe an endocannabinoid-like molecule, oleoyl serotonin (OS), present in the venom of Stephanoconus snails. Using electrophysiological assays, it was shown that OS inhibits CB1 and CB2. Structure–activity relationship studies using a chimeric CB1/2 revealed that the domain encompassing the transmembrane helix V (TMHV)– intracellular loop 3 (ICL3)–TMHVI of the CB2 is critical for the binding and function of OS. We concluded that OS binds to sites of the CB2 that are different from the binding sites of the non-selective CB agonist WIN55,212-2. Behavioral assays in mice showed that OS counteracted learning and memory deficits caused by WIN55,212-2. Furthermore, a selectivity screening of OS showed high selectivity for CB over various ion channels and receptors. Overall, OS may represent a new approach to the prevention and treatment of learning and memory cognition impairment in neurological diseases. Full article

KCNE3 is a single-pass integral membrane protein that regulates numerous voltage-gated potassium channel functions such as KCNQ1. Previous solution NMR studies suggested a moderate degree of curved α-helical structure in the transmembrane domain (TMD) of KCNE3 in lyso-myristoylphosphatidylcholine (LMPC) micelles and isotropic bicelles with the residues T71, S74 and G78 situated along the concave face of the curved helix. During the interaction of KCNE3 and KCNQ1, KCNE3 pushes its transmembrane domain against KCNQ1 to lock the voltage sensor in its depolarized conformation. A cryo-EM study of KCNE3 complexed with KCNQ1 in nanodiscs suggested a deviation of the KCNE3 structure from its independent structure in isotropic bicelles. Despite the biological significance of KCNE3 TMD, the conformational properties of KCNE3 are poorly understood. Here, all atom molecular dynamics (MD) simulations were utilized to investigate the conformational dynamics of the transmembrane domain of KCNE3 in a lipid bilayer containing a mixture of POPC and POPG lipids (3:1). Further, the effect of the interaction impairing mutations (V72A, I76A and F68A) on the conformational properties of the KCNE3 TMD in lipid bilayers was investigated. Our MD simulation results suggest that the KCNE3 TMD adopts a nearly linear α helical structural conformation in POPC-POPG lipid bilayers. Additionally, the results showed no significant change in the nearly linear α-helical conformation of KCNE3 TMD in the presence of interaction impairing mutations within the sampled time frame. The KCNE3 TMD is more stable with lower flexibility in comparison to the N-terminal and C-terminal of KCNE3 in lipid bilayers. The overall conformational flexibility of KCNE3 also varies in the presence of the interaction-impairing mutations. The MD simulation data further suggest that the membrane bilayer width is similar for wild-type KCNE3 and KCNE3 containing mutations. The Z-distance measurement data revealed that the TMD residue site A69 is close to the lipid bilayer center, and residue sites S57 and S82 are close to the surfaces of the lipid bilayer membrane for wild-type KCNE3 and KCNE3 containing interaction-impairing mutations. These results agree with earlier KCNE3 biophysical studies. The results of these MD simulations will provide complementary data to the experimental outcomes of KCNE3 to help understand its conformational dynamic properties in a more native lipid bilayer environment. Full article