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During HIV-1 infection, the envelope glycoprotein subunit gp41 folds into a six-helix bundle structure (6HB) formed by the interaction between its N-terminal (NHR) and C-terminal (CHR) heptad-repeats, promoting viral and cell membranes fusion. A highly preserved, hydrophobic pocket (HP) on the NHR surface is crucial in 6HB formation and, therefore, HP-binding compounds constitute promising therapeutics against HIV-1. Here, we investigated the conformational and dynamic properties of the HP using a rationally designed single-chain protein (named covNHR) that mimics the gp41 NHR structure. We found that the fluorescent dye 8-anilino-naphtalene- 1-sulfonic acid (ANS) binds specifically to the HP, suggesting that ANS derivatives may constitute lead compounds to inhibit 6HB formation. ANS shows different binding modes to the HP, depending on the occupancy of other NHR pockets. Moreover, in presence of a CHR peptide bound to the N-terminal pockets in gp41, two ANS molecules can occupy the HP showing cooperative behavior. This binding mode was assessed using molecular docking and molecular dynamics simulations. The results show that the HP is conformationally flexible and connected allosterically to other NHR regions, which strongly influence the binding of potential ligands. These findings could guide the development of small-molecule HIV-1 inhibitors targeting the HP., Spanish State Research Agency, SRA/ 10.13039/501100011033 (grants BIO2016-76640-R and PID2019.107515RB.C21), ERDF/ESF

Research on the primary cilium has been growing exponentially in the past several decades due to its functions as a cell signalling hub, which defects leads to several disorders and abnormalities collectively known as ciliopathies. Among other parts of the primary cilium structures, the transition zone is the area whose defects lead to the most severe clinical manifestations and high lethality. The ciliary transition zone consists of multiple protein modules that are hypothesized to be anchored by the RPGRIP1L protein. Despite its importance, RPGRIP1L studies remain hidden from the limelight, and our understanding of the protein remains scattered. This review summarizes the clinical manifestations and molecular mechanisms of the RPGRIP1L in the primary cilium. We then take a closer look at each RPGRIP1L’s protein domain to understand how each domain ensures proper functions and localization of RPGRIP1L. The three domains of RPGRIP1L are postulated to be involved in different roles. While the coiled coil domain is vital for scaffolding the protein to the centriolar structure, the ability of the C2 domain to interact with lipid allows the formation of ‘lipid gate’ at the transition zone. The high variability of the RPGR interaction domain enable the RPGRIP1L to interact with multiple different proteins, making it an ideal anchor for other ciliary protein modules in the transition zone.

In bacteria, Lon is a large hexameric ATP-dependent protease that targets misfolded and also folded substrates, some of which are involved in cell division and survival of cellular stress. The N-terminal domain of Lon facilitates substrate recognition, but how the domains confer such activity has remained unclear. Here, we report the full-length structure of Lon protease from Thermus thermophilus at 3.9 Å resolution in a substrate-engaged state. The six N-terminal domains are arranged in three pairs, stabilized by coiled-coil segments and forming an additional channel for substrate sensing and entry into the AAA+ ring. Sequence conservation analysis and proteolysis assays confirm that this architecture is required for the degradation of both folded and unfolded substrates in bacteria.

Surface layer proteins perform multiple functions in prokaryotic cells, including cellular defense, cell-shape maintenance, and regulation of import and export of materials. However, mimicking the complex and dynamic behavior of such two-dimensional biochemical systems is challenging, and hence research has so far focused mainly on the design and manipulation of the structure and functionality of protein assemblies in solution. Motivated by the new opportunities that dynamic surface layer proteins may offer for modern technology, we herein demonstrate that immobilization of coiled coil proteins onto an inorganic surface facilitates complex behavior, manifested by reversible chemical reactions that can be rapidly monitored as digital surface readouts. Using multiple chemical triggers as inputs and several surface characteristics as outputs, we can realize reversible switching and logic gate operations that are read in parallel. Moreover, using the same coiled coil protein monolayers for derivatization of nanopores drilled into silicon nitride membranes facilitates control over ion and mass transport through the pores, thereby expanding the applicability of the dynamic coiled coil system for contemporary stochastic biosensing applications.

Transmembrane receptors are central components of the chemosensory systems by which motile bacteria detect and respond to chemical gradients. An attractant bound to the receptor periplasmic domain generates conformational signals that regulate a histidine kinase interacting with its cytoplasmic domain. Ligand-induced signaling through the periplasmic and transmembrane domains of the receptor involves a piston-like helical displacement, but the nature of this signaling through the >200 A four-helix coiled coil of the cytoplasmic domain had not yet been identified. We performed single-molecule Forster resonance energy transfer measurements on Escherichia coli aspartate receptor homodimers inserted into native phospholipid bilayers enclosed in nanodiscs. The receptors were labeled with fluorophores at diagnostic positions near the middle of the cytoplasmic coiled coil. At these positions, we found that the two N-helices of the homodimer were more distant, that is, less tightly packed and more dynamic than the companion C-helix pair, consistent with previous deductions that the C-helices form a stable scaffold and the N-helices are dynamic. Upon ligand binding, the scaffold pair compacted further, while separation and dynamics of the dynamic pair increased. Thus, ligand binding had asymmetric effects on the two helical pairs, shifting mean distances in opposite directions and increasing the dynamics of one pair. We suggest that this reflects a conformational change in which differential alterations to the packing and dynamics of the two helical pairs are coupled. These coupled changes could represent a previously unappreciated mode of conformational signaling that may well occur in other coiled-coil signaling proteins.

Herein we report unprecedented location‐dependent, size‐selective binding to designed lanthanide (Ln3+) sites within miniature protein coiled coil scaffolds. Not only do these engineered sites display unusual Ln3+ selectivity for moderately large Ln3+ ions (Nd to Tb), for the first time we demonstrate that selectivity can be location‐dependent and can be programmed into the sequence. A 1 nm linear translation of the binding site towards the N‐terminus can convert a selective site into a highly promiscuous one. An X‐ray crystal structure, the first of a lanthanide binding site within a coiled coil to be reported, coupled with CD studies, reveal the existence of an optimal radius that likely stems from the structural constraints of the coiled coil scaffold. To the best of our knowledge this is the first report of location‐dependent metal selectivity within a coiled coil scaffold, as well as the first report of location‐dependent Ln3+ selectivity within a protein., Location‐dependent size selectivity can be programmed into designed metallo coiled coils, allowing for the generation of near‐identical selective and promiscuous metal‐ion sites. The first crystal structure of a lanthanide bound within a coiled coil reveals that the existence of an optimal radius stems from the structural constraints of the peptide scaffold. Therefore, a selective site can in principle be designed de novo for any given Ln3+ ion.

The two-domain actin associated protein coronin interacts with filamentous (F-) actin, facilitating diverse biological processes including cell proliferation, motility, phagocytosis, host-parasite interaction and cargo binding. The conserved N-terminal β-propeller domain is involved in protein: protein interactions, while the C-terminal coiled-coil domain mediates oligomerization, transducing conformational changes. The L. donovani coronin coiled-coil (LdCoroCC) domain exhibited a novel topology and oligomer association with an inherent asymmetry, caused primarily by three a residues of successive heptads. In the T.brucei homolog (TbrCoro), two of these ‘a’ residues are different (Val 493 & 507 replacing LdCoroCC Ile 486 and Met 500 respectively). The elucidated structure possesses a similar topology and assembly while comparative structural analysis shows that the T.brucei coronin coiled-coil domain (TbrCoroCC) too possesses the asymmetry though its magnitude is smaller. Analysis identifies that the asymmetric state is stabilized via cyclic salt bridges formed by Arg 497 and Glu 504. Co-localization studies (LdCoro, TbrCoro and corresponding mutant coiled coil constructs) with actin show that there are subtle differences in their binding patterns, with the double mutant V493I–V507M showing maximal effect. None of the constructs have an effect on F-actin length. Taken together with LdCoroCC, we therefore conclude that the inherent asymmetric structures are essential for kinetoplastids, and are of interest in understanding and exploiting actin dynamics., Graphical abstract Image 1, Highlights • T.brucei coronin coiled-coil domain (TbrCoroCC) is an anti-parallel four-helix bundle as the L.donovani homolog(LdCoroCC). • Magnitude of asymmetry observed in TbrCoroCC is different from that in LdCoroCC. • Cyclic salt bridges stabilize the tetramer with contributions from steric interactions. • LdCoroCC and TbrCoroCC constructs show subtle differences in actin association in vivo.

— SARS-CoV-2, the causative agent of COVID-19, remains the focus of research worldwide. SARS-CoV-2 entry into the cell starts with its S protein binding to the angiotensin-converting enzyme-2 (ACE2) expressed on the cell surface. The knowledge of the S protein’s spatial structure is indispensable for understanding the molecular principles of its work. The S protein structure has been almost fully described using experimental approaches with the only exception for the protein’s endodomain, the transmembrane domain, and the ectodomain parts adjacent to the latter. The paper reports molecular modelling of the S protein fragment corresponding to its coiled coil HR2 domain and fully palmitoylated transmembrane domain. Model stability in lipid bilayer was confirmed by all-atom and coarse-grained molecular dynamics simulations. It has been demonstrated that palmitoylation leads to a significant decrease in transmembrane domain mobility and local bilayer thickening, which may be relevant for protein trimerization.

Copper nitrite reductase (CuNiR) is a copper enzyme that converts nitrite to nitric oxide and is an important part of the global nitrogen cycle in bacteria. The relatively simple CuHis3 binding site of the CuNiR active site has made it an enticing target for small molecule modeling and de novo protein design studies. We have previously reported symmetric CuNiR models within parallel three stranded coiled coil systems, with activities that span a range of three orders of magnitude. In this report, we investigate the same CuHis3 binding site within an antiparallel three helical bundle scaffold, which allows the design of asymmetric constructs. We determine that a simple CuHis3 binding site can be designed within this scaffold with enhanced activity relative to the comparable construct in parallel coiled coils. Incorporating more complex designs or repositioning this binding site can decrease this activity as much as 15 times. Comparing these constructs, we reaffirm a previous result in which a blue shift in the 1s to 4p transition energy determined by Cu(I) X-ray absorption spectroscopy is correlated with an enhanced activity within imidazole-based constructs. With this step and recent successful electron transfer site designs within this scaffold, we are one step closer to a fully functional de novo designed nitrite reductase.

One approach to protein assembly involves water‐soluble supramolecular receptors that act like glues. Bionanoarchitectures directed by these scaffolds are often system‐specific, with few studies investigating their customization. Herein, the modulation of cucurbituril‐mediated protein assemblies through the inclusion of peptide tectons is described. Three peptides of varying length and structural order were N‐terminally appended to RSL, a β‐propeller building block. Each fusion protein was incorporated into crystalline architectures mediated by cucurbit[7]uril (Q7). A trimeric coiled‐coil served as a spacer within a Q7‐directed sheet assembly of RSL, giving rise to a layered material of varying porosity. Within the spacer layers, the coiled‐coils were dynamic. This result prompted consideration of intrinsically disordered peptides (IDPs) as modulatory tectons. Similar to the coiled‐coil, a mussel adhesion peptide (Mefp) also acted as a spacer between protein–Q7 sheets. In contrast, the fusion of a nucleoporin peptide (Nup) to RSL did not recapitulate the sheet assembly. Instead, a Q7‐directed cage was adopted, within which disordered Nup peptides were partially “captured” by Q7 receptors. IDP capture occurred by macrocycle recognition of an intrapeptide Phe‐Gly motif in which the benzyl group was encapsulated by Q7. The modularity of these protein–cucurbituril architectures adds a new dimension to macrocycle‐mediated protein assembly. Segregated protein crystals, with alternating layers of high and low porosity, could provide a basis for new types of materials., Bio‐supramolecular tectonics. The bionanoarchitectures of a β‐propeller protein and cucurbit[7]uril (Q7) were customized by incorporating peptide tectons. Segregated crystalline assemblies with alternating rigid and flexible layers were obtained. The flexible spacer layer could be achieved with either structured (coiled‐coil) or disordered (mussel foot peptide) tectons fused to the β‐propeller protein. A protein–Q7 cage‐like architecture partially captured a disordered nucleoporin peptide.

The protein stromal interaction molecule 1 (STIM1) plays a pivotal role in mediating store-operated calcium entry (SOCE) into cells, which is essential for adaptive immunity. It acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol, where it changes from an inactive (tight) to an active (extended) oligomeric form upon calcium store depletion. NMR studies of this protein are challenging due to its membrane-spanning and aggregation properties. Therefore follow the divide-and-conquer approach, focusing on individual domains first is in order. The cytosolic part is predicted to have a large content of coiled-coil (CC) structure. We report the 1H, 13C, 15N chemical shift assignments of the CC3 domain. This domain is crucial for the stabilisation of the tight quiescent form of STIM1 as well as for activating the ORAI calcium channel by direct contact, in the extended active form. Supplementary Information The online version contains supplementary material available at 10.1007/s12104-021-10042-7.

Coiled-coil domain-containing 124 (CCDC124) is a recently discovered ribosome-binding protein conserved in eukaryotes. CCDC124 has regulatory functions on the mediation of reversible ribosomal hibernation and translational recovery by direct attachment to large subunit ribosomal protein uL5, 25S rRNA backbone, and tRNA binding P/A-site major groove. Moreover, it independently mediates cell division and cellular stress response by facilitating cytokinetic abscission and disulfide stress-dependent transcriptional regulation, respectively. However, the structural characterization and intracellular physiological status of CCDC124 remain unknown. In this study, we employed advanced in silico protein modeling and characterization tools to generate a native-like tertiary structure of CCDC124 and examine the disorder, low sequence complexity, and aggregation propensities, as well as high-order dimeric/oligomeric states. Subsequently, dimerization of CCDC124 was investigated with co-immunoprecipitation (CO-IP) analysis, immunostaining, and a recent live-cell protein-protein interaction method, bimolecular fluorescence complementation (BiFC). Results revealed CCDC124 as a highly disordered protein consisting of low complexity regions at the N-terminus and an aggregation sequence (151-IAVLSV-156) located in the middle region. Molecular docking and post-docking binding free energy analyses highlighted a potential involvement of V153 residue on the generation of high-order dimeric/oligomeric structures. Co-IP, immunostaining, and BiFC analyses were used to further confirm the dimeric state of CCDC124 predominantly localized at the cytoplasm. In conclusion, our findings revealed in silico structural characterization and in vivo subcellular physiological state of CCDC124, suggesting low complexity regions located at the N-terminus of disordered CCDC124 may regulate the formation of aggregates or high-order dimeric/oligomeric states. This article is protected by copyright. All rights reserved.

TMEM120A (Transmembrane protein 120A) was recently identified as a mechanical pain sensing ion channel named as TACAN, while its homologue TMEM120B has no mechanosensing property1. Here, we report the cryo-EM structures of both human TMEM120A and TMEM120B. The two structures share the same dimeric assembly, mediated by extensive interactions through the transmembrane domain (TMD) and the N-terminal coiled coil domain (CCD). However, the nearly identical structures cannot provide clues for the difference in mechanosensing between TMEM120A and TMEM120B. Although TMEM120A could mediate conducting currents in a bilayer system, it does not mediate mechanical-induced currents in a heterologous expression system, suggesting TMEM120A is unlikely a mechanosensing channel. Instead, the TMDs of TMEM120A and TMEM120B resemble the structure of a fatty acid elongase, ELOVL7, indicating their potential role of an enzyme in lipid metabolism.

The atomic structure of the complete myosin tail within thick filaments isolated from Lethocerus indicus flight muscle is described and compared to crystal structures of recombinant, human cardiac myosin tail segments. Overall, the agreement is good with three exceptions: the proximal S2, in which the filament has heads attached but the crystal structure doesn’t, and skip regions 2 and 4. At the head–tail junction, the tail α-helices are asymmetrically structured encompassing well-defined unfolding of 12 residues for one myosin tail, ∼4 residues of the other, and different degrees of α-helix unwinding for both tail α-helices, thereby providing an atomic resolution description of coiled-coil “uncoiling” at the head–tail junction. Asymmetry is observed in the nonhelical C termini; one C-terminal segment is intercalated between ribbons of myosin tails, the other apparently terminating at Skip 4 of another myosin tail. Between skip residues, crystal and filament structures agree well. Skips 1 and 3 also agree well and show the expected α-helix unwinding and coiled-coil untwisting in response to skip residue insertion. Skips 2 and 4 are different. Skip 2 is accommodated in an unusual manner through an increase in α-helix radius and corresponding reduction in rise/residue. Skip 4 remains helical in one chain, with the other chain unfolded, apparently influenced by the acidic myosin C terminus. The atomic model may shed some light on thick filament mechanosensing and is a step in understanding the complex roles that thick filaments of all species undergo during muscle contraction.