Visualizing the disordered nuclear transport machinery in situ.

The approximately 120 MDa mammalian nuclear pore complex (NPC) acts as a gatekeeper for the transport between the nucleus and cytosol1. The central channel of the NPC is filled with hundreds of intrinsically disordered proteins (IDPs) called FG-nucleoporins (FG-NUPs)2,3. Although the structure of the NPC scaffold has been resolved in remarkable detail, the actual transport machinery built up by FG-NUPs-about 50 MDa-is depicted as an approximately 60-nm hole in even highly resolved tomograms and/or structures computed with artificial intelligence4-11. Here we directly probed conformations of the vital FG-NUP98 inside NPCs in live cells and in permeabilized cells with an intact transport machinery by using a synthetic biology-enabled site-specific small-molecule labelling approach paired with highly time-resolved fluorescence microscopy. Single permeabilized cell measurements of the distance distribution of FG-NUP98 segments combined with coarse-grained molecular simulations of the NPC allowed us to map the uncharted molecular environment inside the nanosized transport channel. We determined that the channel provides-in the terminology of the Flory polymer theory12-a 'good solvent' environment. This enables the FG domain to adopt expanded conformations and thus control transport between the nucleus and cytoplasm. With more than 30% of the proteome being formed from IDPs, our study opens a window into resolving disorder-function relationships of IDPs in situ, which are important in various processes, such as cellular signalling, phase separation, ageing and viral entry.

Phosphorylation of the receptor protein Pex5p modulates import of proteins into peroxisomes.

Peroxisomes are organelles with vital functions in metabolism and their dysfunction is associated with human diseases. To fulfill their multiple roles, peroxisomes import nuclear-encoded matrix proteins, most carrying a peroxisomal targeting signal (PTS) 1. The receptor Pex5p recruits PTS1-proteins for import into peroxisomes; whether and how this process is posttranslationally regulated is unknown. Here, we identify 22 phosphorylation sites of Pex5p. Yeast cells expressing phospho-mimicking Pex5p-S507/523D (Pex5p2D) show decreased import of GFP with a PTS1. We show that the binding affinity between a PTS1-protein and Pex5p2D is reduced. An in vivo analysis of the effect of the phospho-mimicking mutant on PTS1-proteins revealed that import of most, but not all, cargos is affected. The physiological effect of the phosphomimetic mutations correlates with the binding affinity of the corresponding extended PTS1-sequences. Thus, we report a novel Pex5p phosphorylation-dependent mechanism for regulating PTS1-protein import into peroxisomes. In a broader view, this suggests that posttranslational modifications can function in fine-tuning the peroxisomal protein composition and, thus, cellular metabolism.

Mechanism of conditional partner selectivity in MITF/TFE family transcription factors with a conserved coiled coil stammer motif.

Interrupted dimeric coiled coil segments are found in a broad range of proteins and generally confer selective functional properties such as binding to specific ligands. However, there is only one documented case of a basic-helix-loop-helix leucine zipper transcription factor-microphthalmia-associated transcription factor (MITF)-in which an insertion of a three-residue stammer serves as a determinant of conditional partner selectivity. To unravel the molecular principles of this selectivity, we have analyzed the high-resolution structures of stammer-containing MITF and an engineered stammer-less MITF variant, which comprises an uninterrupted symmetric coiled coil. Despite this fundamental difference, both MITF structures reveal identical flanking in-phase coiled coil arrangements, gained by helical over-winding and local asymmetry in wild-type MITF across the stammer region. These conserved structural properties allow the maintenance of a proper functional readout in terms of nuclear localization and binding to specific DNA-response motifs regardless of the presence of the stammer. By contrast, MITF heterodimer formation with other bHLH-Zip transcription factors is only permissive when both factors contain either the same type of inserted stammer or no insert. Our data illustrate a unique principle of conditional partner selectivity within the wide arsenal of transcription factors with specific partner-dependent functional readouts.

ColBuilder: A server to build collagen fibril models.

Type I collagen is the main structural component of many tissues in the human body. It provides excellent mechanical properties to connective tissue and acts as a protein interaction hub. There is thus a wide interest in understanding the properties and diverse functions of type I collagen at the molecular level. A precondition is an atomistic collagen I structure as it occurs in native tissue. To this end, we built full-atom models of cross-linked collagen fibrils by integrating the low-resolution structure of collagen fibril available from x-ray fiber diffraction with high-resolution structures of short collagen-like peptides from x-ray crystallography and mass spectrometry data. We created a Web resource of collagen models for 20 different species with a large variety of cross-link types and localization within the fibril to facilitate structure-based analyses and simulations of type I collagen in health and disease. To easily enable simulations, we provide parameters of the modeled cross-links for an Amber force field. The repository of collagen models is available at https://colbuilder.h-its.org.

Structure and operation of the DNA-translocating type I DNA restriction enzymes.

Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes.

PepComposer: computational design of peptides binding to a given protein surface.

There is a wide interest in designing peptides able to bind to a specific region of a protein with the aim of interfering with a known interaction or as starting point for the design of inhibitors. Here we describe PepComposer, a new pipeline for the computational design of peptides binding to a given protein surface. PepComposer only requires the target protein structure and an approximate definition of the binding site as input. We first retrieve a set of peptide backbone scaffolds from monomeric proteins that harbor the same backbone arrangement as the binding site of the protein of interest. Next, we design optimal sequences for the identified peptide scaffolds. The method is fully automatic and available as a web server at http://biocomputing.it/pepcomposer/webserver.

The palisade layer of the poxvirus core is composed of flexible A10 trimers.

Due to its asymmetric shape, size and compactness, the structure of the infectious mature virus (MV) of vaccinia virus (VACV), the best-studied poxvirus, remains poorly understood. Instead, subviral particles, in particular membrane-free viral cores, have been studied with cryo-electron microscopy. Here, we compared viral cores obtained by detergent stripping of MVs with cores in the cellular cytoplasm, early in infection. We focused on the prominent palisade layer on the core surface, combining cryo-electron tomography, subtomogram averaging and AlphaFold2 structure prediction. We showed that the palisade is composed of densely packed trimers of the major core protein A10. Trimers display a random order and their classification indicates structural flexibility. A10 on cytoplasmic cores is organized in a similar manner, indicating that the structures obtained in vitro are physiologically relevant. We discuss our results in the context of the VACV replicative cycle, and the assembly and disassembly of the infectious MV.

Co-translational binding of importins to nascent proteins.

Various cellular quality control mechanisms support proteostasis. While, ribosome-associated chaperones prevent the misfolding of nascent chains during translation, importins were shown to prevent the aggregation of specific cargoes in a post-translational mechanism prior the import into the nucleoplasm. Here, we hypothesize that importins may already bind ribosome-associated cargo in a co-translational manner. We systematically measure the nascent chain association of all importins in Saccharomyces cerevisiae by selective ribosome profiling. We identify a subset of importins that bind to a wide range of nascent, often uncharacterized cargoes. This includes ribosomal proteins, chromatin remodelers and RNA binding proteins that are aggregation prone in the cytosol. We show that importins act consecutively with other ribosome-associated chaperones. Thus, the nuclear import system is directly intertwined with nascent chain folding and chaperoning.

AI-based structure prediction empowers integrative structural analysis of human nuclear pores.

INTRODUCTION The eukaryotic nucleus pro-tects the genome and is enclosed by the two membranes of the nuclear envelope. Nuclear pore complexes (NPCs) perforate the nuclear envelope to facilitate nucleocytoplasmic transport. With a molecular weight of ∼120 MDa, the human NPC is one of the larg-est protein complexes. Its ~1000 proteins are taken in multiple copies from a set of about 30 distinct nucleoporins (NUPs). They can be roughly categorized into two classes. Scaf-fold NUPs contain folded domains and form a cylindrical scaffold architecture around a central channel. Intrinsically disordered NUPs line the scaffold and extend into the central channel, where they interact with cargo complexes. The NPC architecture is highly dynamic. It responds to changes in nuclear envelope tension with conforma-tional breathing that manifests in dilation and constriction movements. Elucidating the scaffold architecture, ultimately at atomic resolution, will be important for gaining a more precise understanding of NPC function and dynamics but imposes a substantial chal-lenge for structural biologists. RATIONALE Considerable progress has been made toward this goal by a joint effort in the field. A synergistic combination of complementary approaches has turned out to be critical. In situ structural biology techniques were used to reveal the overall layout of the NPC scaffold that defines the spatial reference for molecular modeling. High-resolution structures of many NUPs were determined in vitro. Proteomic analysis and extensive biochemical work unraveled the interaction network of NUPs. Integra-tive modeling has been used to combine the different types of data, resulting in a rough outline of the NPC scaffold. Previous struc-tural models of the human NPC, however, were patchy and limited in accuracy owing to several challenges: (i) Many of the high-resolution structures of individual NUPs have been solved from distantly related species and, consequently, do not comprehensively cover their human counterparts. (ii) The scaf-fold is interconnected by a set of intrinsically disordered linker NUPs that are not straight-forwardly accessible to common structural biology techniques. (iii) The NPC scaffold intimately embraces the fused inner and outer nuclear membranes in a distinctive topol-ogy and cannot be studied in isolation. (iv) The conformational dynamics of scaffold NUPs limits the resolution achievable in structure determination. RESULTS In this study, we used artificial intelligence (AI)-based prediction to generate an exten-sive repertoire of structural models of human NUPs and their subcomplexes. The resulting models cover various domains and interfaces that so far remained structurally uncharac-terized. Benchmarking against previous and unpublished x-ray and cryo-electron micros-copy structures revealed unprecedented accu-racy. We obtained well-resolved cryo-electron tomographic maps of both the constricted and dilated conformational states of the hu-man NPC. Using integrative modeling, we fit-ted the structural models of individual NUPs into the cryo-electron microscopy maps. We explicitly included several linker NUPs and traced their trajectory through the NPC scaf-fold. We elucidated in great detail how mem-brane-associated and transmembrane NUPs are distributed across the fusion topology of both nuclear membranes. The resulting architectural model increases the structural coverage of the human NPC scaffold by about twofold. We extensively validated our model against both earlier and new experimental data. The completeness of our model has enabled microsecond-long coarse-grained molecular dynamics simulations of the NPC scaffold within an explicit membrane en-vironment and solvent. These simulations reveal that the NPC scaffold prevents the constriction of the otherwise stable double-membrane fusion pore to small diameters in the absence of membrane tension. CONCLUSION Our 70-MDa atomically re-solved model covers >90% of the human NPC scaffold. It captures conforma-tional changes that occur during dilation and constriction. It also reveals the precise anchoring sites for intrinsically disordered NUPs, the identification of which is a prerequisite for a complete and dy-namic model of the NPC. Our study exempli-fies how AI-based structure prediction may accelerate the elucidation of subcellular ar-chitecture at atomic resolution. [Figure: see text].

The structure of M.EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein.

Type-I DNA restriction-modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of DNA or translocation and cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein ocr, a DNA mimic. We present a 3D density map generated by negative-stain electron microscopy and single particle analysis of the central core of the restriction complex, the M.EcoKI M(2)S(1) methyltransferase, bound to ocr. We also present complete atomic models of M.EcoKI in complex with ocr and its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of experimental data on EcoKI published over 40 years.

Nuclear pores dilate and constrict in cellulo.

In eukaryotic cells, nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes and mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins and form a cylindrical architecture around an aqueous central channel. This architecture is highly dynamic in space and time. Variations in NPC diameter have been reported, but the physiological circumstances and the molecular details remain unknown. Here, we combined cryo­electron tomography with integrative structural modeling to capture a molecular movie of the respective large-scale conformational changes in cellulo. Although NPCs of exponentially growing cells adopted a dilated conformation, they reversibly constricted upon cellular energy depletion or conditions of hypertonic osmotic stress. Our data point to a model where the nuclear envelope membrane tension is linked to the conformation of the NPC.

Hybrid Kinetic Monte Carlo/Molecular Dynamics Simulations of Bond Scissions in Proteins.

Proteins are exposed to various mechanical loads that can lead to covalent bond scissions even before macroscopic failure occurs. Knowledge of these molecular breakages is important to understand mechanical properties of the protein. In regular molecular dynamics (MD) simulations, covalent bonds are predefined, and reactions cannot occur. Furthermore, such events rarely take place on MD time scales. Existing approaches that tackle this limitation either rely on computationally expensive quantum calculations (e.g., QM/MM) or complex bond order formalisms in force fields (e.g., ReaxFF). To circumvent these limitations, we present a new reactive kinetic Monte Carlo/molecular dynamics (KIMMDY) scheme. Here, bond rupture rates are calculated based on the interatomic distances in the MD simulation and then serve as an input for a kinetic Monte Carlo step. This easily scalable hybrid approach drastically increases the accessible time scales. Using this new technique, we investigate bond ruptures in a multimillion atom system of tensed collagen, a structural protein found in skin, bones, and tendons. Our findings show a clear concentration of bond scissions near chemical cross-links in collagen. We also examine subsequent dynamic relaxation steps. Our method exhibits only a minor slowdown compared to classical MD and is straightforwardly applicable to other complex (bio)materials under load and related chemistries.

Mechanoradicals in tensed tendon collagen as a source of oxidative stress.

As established nearly a century ago, mechanoradicals originate from homolytic bond scission in polymers. The existence, nature and biological relevance of mechanoradicals in proteins, instead, are unknown. We here show that mechanical stress on collagen produces radicals and subsequently reactive oxygen species, essential biological signaling molecules. Electron-paramagnetic resonance (EPR) spectroscopy of stretched rat tail tendon, atomistic molecular dynamics simulations and quantum-chemical calculations show that the radicals form by bond scission in the direct vicinity of crosslinks in collagen. Radicals migrate to adjacent clusters of aromatic residues and stabilize on oxidized tyrosyl radicals, giving rise to a distinct EPR spectrum consistent with a stable dihydroxyphenylalanine (DOPA) radical. The protein mechanoradicals, as a yet undiscovered source of oxidative stress, finally convert into hydrogen peroxide. Our study suggests collagen I to have evolved as a radical sponge against mechano-oxidative damage and proposes a mechanism for exercise-induced oxidative stress and redox-mediated pathophysiological processes.

Functional analysis of MmeI from methanol utilizer Methylophilus methylotrophus, a subtype IIC restriction-modification enzyme related to type I enzymes.

MmeI from Methylophilus methylotrophus belongs to the type II restriction-modification enzymes. It recognizes an asymmetric DNA sequence, 5'-TCCRAC-3' (R indicates G or A), and cuts both strands at fixed positions downstream of the specific site. This particular feature has been exploited in transcript profiling of complex genomes (using serial analysis of gene expression technology). We have shown previously that the endonucleolytic activity of MmeI is strongly dependent on the presence of S-adenosyl-l-methionine (J. Nakonieczna, J. W. Zmijewski, B. Banecki, and A. J. Podhajska, Mol. Biotechnol. 37:127-135, 2007), which puts MmeI in subtype IIG. The same cofactor is used by MmeI as a methyl group donor for modification of an adenine in the upper strand of the recognition site to N(6)-methyladenine. Both enzymatic activities reside in a single polypeptide (919 amino acids [aa]), which puts MmeI also in subtype IIC of the restriction-modification systems. Based on a molecular model, generated with the use of bioinformatic tools and validated by site-directed mutagenesis, we were able to localize three functional domains in the structure of the MmeI enzyme: (i) the N-terminal portion containing the endonucleolytic domain with the catalytic Mg2+-binding motif D(70)-X(9)-EXK(82), characteristic for the PD-(D/E)XK superfamily of nucleases; (ii) a central portion (aa 310 to 610) containing nine sequence motifs conserved among N(6)-adenine gamma-class DNA methyltransferases; (iii) the C-terminal portion (aa 610 to 919) containing a putative target recognition domain. Interestingly, all three domains showed highest similarity to the corresponding elements of type I enzymes rather than to classical type II enzymes. We have found that MmeI variants deficient in restriction activity (D70A, E80A, and K82A) can bind and methylate specific nucleotide sequence. This suggests that domains of MmeI responsible for DNA restriction and modification can act independently. Moreover, we have shown that a single amino acid residue substitution within the putative target recognition domain (S807A) resulted in a MmeI variant with a higher endonucleolytic activity than the wild-type enzyme.

HsdR subunit of the type I restriction-modification enzyme EcoR124I: biophysical characterisation and structural modelling.

Type I restriction-modification (RM) systems are large, multifunctional enzymes composed of three different subunits. HsdS and HsdM form a complex in which HsdS recognizes the target DNA sequence, and HsdM carries out methylation of adenosine residues. The HsdR subunit, when associated with the HsdS-HsdM complex, translocates DNA in an ATP-dependent process and cleaves unmethylated DNA at a distance of several thousand base-pairs from the recognition site. The molecular mechanism by which these enzymes translocate the DNA is not fully understood, in part because of the absence of crystal structures. To date, crystal structures have been determined for the individual HsdS and HsdM subunits and models have been built for the HsdM-HsdS complex with the DNA. However, no structure is available for the HsdR subunit. In this work, the gene coding for the HsdR subunit of EcoR124I was re-sequenced, which showed that there was an error in the published sequence. This changed the position of the stop codon and altered the last 17 amino acid residues of the protein sequence. An improved purification procedure was developed to enable HsdR to be purified efficiently for biophysical and structural analysis. Analytical ultracentrifugation shows that HsdR is monomeric in solution, and the frictional ratio of 1.21 indicates that the subunit is globular and fairly compact. Small angle neutron-scattering of the HsdR subunit indicates a radius of gyration of 3.4 nm and a maximum dimension of 10 nm. We constructed a model of the HsdR using protein fold-recognition and homology modelling to model individual domains, and small-angle neutron scattering data as restraints to combine them into a single molecule. The model reveals an ellipsoidal shape of the enzymatic core comprising the N-terminal and central domains, and suggests conformational heterogeneity of the C-terminal region implicated in binding of HsdR to the HsdS-HsdM complex.