Intermediate filaments (IFs) are major constituents of the cytoskeleton and nuclear boundary in animal cells. They are of prime importance for the functional organization of structural elements. ...Depending on the cell type, morphologically similar but biochemically distinct proteins form highly viscoelastic filament networks with multiple nanomechanical functions. Besides their primary role in cell plasticity and their established function as cellular stress absorbers, recently discovered gene defects have elucidated that structural alterations of IFs can affect their involvement both in signaling and in controlling gene regulatory networks. Here, we highlight the basic structural and functional properties of IFs and derive a concept of how mutations may affect cellular architecture and thereby tissue construction and physiology.
Abstract
Motivation
Accurate molecular structure of the protein dimer representing the elementary building block of intermediate filaments (IFs) is essential towards the understanding of the filament ...assembly, rationalizing their mechanical properties and explaining the effect of disease-related IF mutations. The dimer contains a ∼300-residue long α-helical coiled coil which cannot be assessed by either direct experimental structure determination or modelling using standard approaches. At the same time, coiled coils are well-represented in structural databases.
Results
Here we present CCFold, a generally applicable threading-based algorithm which produces coiled-coil models from protein sequence only. The algorithm is based on a statistical analysis of experimentally determined structures and can handle any hydrophobic repeat patterns in addition to the most common heptads. We demonstrate that CCFold outperforms general-purpose computational folding in terms of accuracy, while being faster by orders of magnitude. By combining the CCFold algorithm and Rosetta folding we generate representative dimer models for all IF protein classes.
Availability and implementation
The source code is freely available at https://github.com/biocryst/IF; a web server to run the program is at http://pharm.kuleuven.be/Biocrystallography/cc.
Supplementary information
Supplementary data are available at Bioinformatics online.
Intermediate filaments (IFs) result from a key cytoskeletal protein class in metazoan cells, but currently there is no consensus as to their three-dimensional architecture. IF proteins form elongated ...dimers based on the coiled-coil structure within their central ‘rod’ domain. Here we focus on the atomic structure of this elementary dimer, elucidated using X-ray crystallography on multiple fragments and electron paramagnetic resonance experiments on spin-labelled vimentin samples. In line with conserved sequence features, the rod of all IF proteins is composed of three coiled-coil segments containing heptad and hendecad repeats and interconnected by linkers. In addition, the next assembly intermediate beyond the dimer, the tetramer, could be modelled. The impact of these structural results towards understanding the assembly mechanism is discussed.
Together with actin filaments and microtubules, intermediate filaments (IFs) are the basic cytoskeletal components of metazoan cells. Over 80 human diseases have been linked to mutations in various ...IF proteins to date. However, the filament structure is far from being resolved at the atomic level, which hampers rational understanding of IF pathologies. The elementary building block of all IF proteins is a dimer consisting of an α-helical coiled-coil (CC) "rod" domain flanked by the flexible head and tail domains. Here we present three crystal structures of overlapping human vimentin fragments that comprise the first half of its rod domain. Given the previously solved fragments, a nearly complete atomic structure of the vimentin rod has become available. It consists of three α-helical segments (coils 1A, 1B, and 2) interconnected by linkers (L1 and L12). Most of the CC structure has a left-handed twist with heptad repeats, but both coil 1B and coil 2 also exhibit untwisted, parallel stretches with hendecad repeats. In the crystal structure, linker L1 was found to be α-helical without being involved in the CC formation. The available data allow us to construct an atomic model of the antiparallel tetramer representing the second level of vimentin assembly. Although the presence of the nonhelical head domains is essential for proper tetramer stabilization, the precise alignment of the dimers forming the tetramer appears to depend on the complementarity of their surface charge distribution patterns, while the structural plasticity of linker L1 and coil 1A plays a role in the subsequent IF assembly process.
Intermediate filaments (IFs) constitute a major structural element of animal cells. They build two distinct systems, one in the nucleus and one in the cytoplasm. In both cases, their major function ...is assumed to be that of a mechanical stress absorber and an integrating device for the entire cytoskeleton. In line with this, recent disease mutations in human IF proteins indicate that the nanomechanical properties of cell-type-specific IFs are central to the pathogenesis of diseases as diverse as muscular dystrophy and premature ageing. However, the analysis of these various diseases suggests that IFs also have an important role in cell-type-specific physiological functions.
α-Helical coiled coils (CCs) represent an important, highly regular protein folding motif. To date, many thousands of CC structures have been determined experimentally. Their geometry is usually ...modelled by theoretical equations introduced by F. Crick that involve a predefined set of parameters. Here we have addressed the problem of efficient CC parameterization from scratch by performing a statistical evaluation of all available CC structures. The procedure is based on the principal component analysis and yields a minimal set of independent parameters that provide for the reconstruction of the complete CC structure at a required precision. The approach is successfully validated on a set of canonical parallel CC dimers. Its applications include all cases where an efficient sampling of the CC geometry is important, such as for solving the phase problem in crystallography.
Lens epithelium-derived growth factor (LEDGF/p75) is a cellular cofactor of HIV-1 integrase that promotes viral integration by tethering the preintegration complex to the chromatin. By virtue of its ...crucial role in the early steps of HIV replication, the interaction between LEDGF/p75 and integrase represents an attractive target for antiviral therapy. We have rationally designed a series of 2-(quinolin-3-yl)acetic acid derivatives (LEDGINs) that act as potent inhibitors of the LEDGF/p75-integrase interaction and HIV-1 replication at submicromolar concentration by blocking the integration step. A 1.84-A resolution crystal structure corroborates the binding of the inhibitor in the LEDGF/p75-binding pocket of integrase. Together with the lack of cross-resistance with two clinical integrase inhibitors, these findings define the 2-(quinolin-3-yl)acetic acid derivatives as the first genuine allosteric HIV-1 integrase inhibitors. Our work demonstrates the feasibility of rational design of small molecules inhibiting the protein-protein interaction between a viral protein and a cellular host factor.
Human cytosolic prolyl-tRNA synthetase (HcProRS) catalyses the formation of the prolyl-tRNAPro, playing an important role in protein synthesis. Inhibition of HcProRS activity has been shown to have ...potential benefits in the treatment of fibrosis, autoimmune diseases and cancer. Recently, potent pyrazinamide-based inhibitors were identified by a high-throughput screening (HTS) method, but no further elaboration was reported. The pyrazinamide core is a bioactive fragment found in numerous clinically validated drugs and has been subjected to various modifications. Therefore, we applied a virtual screening protocol to our in-house library of pyrazinamide-containing small molecules, searching for potential novel HcProRS inhibitors. We identified a series of 3-benzylaminopyrazine-2-carboxamide derivatives as positive hits. Five of them were confirmed by a thermal shift assay (TSA) with the best compounds 3b and 3c showing EC50 values of 3.77 and 7.34 µM, respectively, in the presence of 1 mM of proline (Pro) and 3.45 µM enzyme concentration. Co-crystal structures of HcProRS in complex with these compounds and Pro confirmed the initial docking studies and show how the Pro facilitates binding of the ligands that compete with ATP substrate. Modelling 3b into other human class II aminoacyl-tRNA synthetases (aaRSs) indicated that the subtle differences in the ATP binding site of these enzymes likely contribute to its potential selective binding of HcProRS. Taken together, this study successfully identified novel HcProRS binders from our anti-tuberculosis in-house compound library, displaying opportunities for repurposing old drug candidates for new applications such as therapeutics in HcProRS-related diseases.
Plasminogen activator inhibitor-1 (PAI-1) is the main physiological inhibitor of tissue-type (tPA) and urokinase-type (uPA) plasminogen activators (PAs). Apart from being critically involved in ...fibrinolysis and wound healing, emerging evidence indicates that PAI-1 plays an important role in many diseases, including cardiovascular disease, tissue fibrosis, and cancer. Targeting PAI-1 is therefore a promising therapeutic strategy in PAI-1 related pathologies. Despite ongoing efforts no PAI-1 inhibitors were approved to date for therapeutic use in humans. A better understanding of the molecular mechanisms of PAI-1 inhibition is therefore necessary to guide the rational design of PAI-1 modulators. Here, we present a 1.9 Å crystal structure of PAI-1 in complex with an inhibitory nanobody VHH-s-a93 (Nb93). Structural analysis in combination with biochemical characterization reveals that Nb93 directly interferes with PAI-1/PA complex formation and stabilizes the active conformation of the PAI-1 molecule.
Ubiquitously expressed human small heat shock proteins (sHsps) HspB1, HspB5, HspB6 and HspB8 contain a conserved motif (S/G)RLFD in their N-terminal domain. For each of them, we prepared mutants with ...a replacement of the conserved R by A (R/A mutants) and a complete deletion of the pentapeptide (Δ mutants) and analyzed their heterooligomerization with other wild-type (WT) human sHsps. We found that WT HspB1 and HspB5 formed heterooligomers with HspB6 only upon heating. In contrast, both HspB1 mutants interacted with WT HspB6 even at low temperature. HspB1/HspB6 heterooligomers revealed a broad size distribution with equimolar ratio suggestive of heterodimers as building blocks, while HspB5/HspB6 heterooligomers had an approximate 2:1 ratio. In contrast, R/A or Δ mutants of HspB6, when mixed with either HspB1 or HspB5, resulted in heterooligomers with a highly variable molar ratio and a decreased HspB6 incorporation. No heterooligomerization of HspB8 or its mutants with either HspB1 or HspB5 could be detected. Finally, R/A or Δ mutations had no effect on heterooligomerization of HspB1 and HspB5 as analyzed by ion exchange chromatography. We conclude that the conserved N-terminal motif plays an important role in heterooligomer formation, as especially pronounced in HspB6 lacking the C-terminal IXI motif.
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