FtsZ filament structures in different nucleotide states reveal the mechanism of assembly dynamics

Treadmilling protein filaments perform essential cellular functions by growing from one end while shrinking from the other, driven by nucleotide hydrolysis. Bacterial cell division relies on the primitive tubulin homolog FtsZ, a target for antibiotic discovery that assembles into single treadmilling filaments that hydrolyse GTP at an active site formed upon subunit association. We determined high-resolution filament structures of FtsZ from the pathogen Staphylococcus aureus in complex with different nucleotide analogs and cations, including mimetics of the ground and transition states of catalysis. Together with mutational and biochemical analyses, our structures reveal interactions made by the GTP γ-phosphate and Mg²⁺ at the subunit interface, a K⁺ ion stabilizing loop T7 for co-catalysis, new roles of key residues at the active site and a nearby crosstalk area, and rearrangements of a dynamic water shell bridging adjacent subunits upon GTP hydrolysis. We propose a mechanistic model that integrates nucleotide hydrolysis signaling with assembly-associated conformational changes and filament treadmilling. Equivalent assembly mechanisms may apply to more complex tubulin and actin cytomotive filaments that share analogous features with FtsZ.

Bacterial cell division critically relies on the tubulin homolog FtsZ, which assembles into filaments that treadmill, fuelled by GTP hydrolysis. This structural and biochemical study of FtsZ from Staphylocuccus aureus reveals the mechanism of GTP hydrolysis and its connection with filament dynamics.     

Analysis of the in vivo assembly pathway of eukaryotic 40S ribosomal proteins

In eukaryotes, in vivo formation of the two ribosomal subunits from four ribosomal RNAs (rRNAs) and approximately 80 ribosomal proteins (r-proteins) involves more than 150 nonribosomal proteins and around 100 small noncoding RNAs. It is temporally and spatially organized within different cellular compartments: the nucleolus, the nucleoplasm, and the cytoplasm. Here, we present a way to analyze how eukaryotic r-proteins of the small ribosomal subunit (SSU) assemble in vivo with rRNA. Our results show that key aspects of the assembly of eukaryotic r-proteins into distinct structural parts of the SSU are similar to the in vitro assembly pathway of their prokaryotic counterparts. We observe that the establishment of a stable assembly intermediate of the eukaryotic SSU body, but not of the SSU head, is closely linked to early rRNA processing events. The formation of assembly intermediates of the head controls efficient nuclear export of the SSU and cytoplasmic pre-rRNA maturation steps.     

Snapshots during the catalytic cycle of a histidine acid phytase reveal an induced-fit structural mechanism

Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P-fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, these data provide important new insights to the factors determining stability, substrate recognition, and the structural mechanism of hydrolysis in this industrially important group of enzymes.     

Structural flexibility of the nucleosome core particle at atomic resolution studied by molecular dynamics simulation

Comparative explicit solvent molecular dynamics (MD) simulations have been performed on a complete nucleosome core particle with and without N-terminal histone tails for more than 20 ns. Main purpose of the simulations was to study the dynamics of mobile elements such as histone N-terminal tails and how packing and DNA-bending influences the fine structure and dynamics of DNA. Except for the tails, histone and DNA molecules stayed on average close to the crystallographic start structure supporting the quality of the current force field approach. Despite the packing strain, no increase of transitions to noncanonical nucleic acid backbone conformations compared to regular B-DNA was observed. The pattern of kinks and bends along the DNA remained close to the experiment overall. In addition to the local dynamics, the simulations allowed the analysis of the superhelical mobility indicating a limited relative mobility of DNA segments separated by one superhelical turn (mean relative displacement of approximately +/-0.2 nm, mainly along the superhelical axis). An even higher rigidity was found for relative motions (distance fluctuations) of segments separated by half a superhelical turn (approximately +/-0.1 nm). The N-terminal tails underwent dramatic conformational rearrangements on the nanosecond time scale toward partially and transiently wrapped states around the DNA. Many of the histone tail changes corresponded to coupled association and folding events from fully solvent-exposed states toward complexes with the major and minor grooves of DNA. The simulations indicate that the rapid conformational changes of the tails can modulate the DNA accessibility within a few nanoseconds.     

State transitions reveal the dynamics and flexibility of the photosynthetic apparatus.

The chloroplast-based photosynthetic apparatus of plants and algae associates various redox cofactors and pigments with approximately 70 polypeptides to form five major transmembrane protein complexes. Among these are two photosystems that have distinct light absorption properties but work in series to produce reducing equivalents aimed at the fixation of atmospheric carbon. A short term chromatic adaptation known as 'State transitions' was discovered thirty years ago that allows photosynthetic organisms to adapt to changes in light quality and intensity which would otherwise compromise the efficiency of photosynthetic energy conversion. A two-decade research effort has finally unraveled the major aspects of the molecular mechanism responsible for State transitions, and their physiological significance has been revisited. This review describes how a-still elusive-regulatory kinase senses the physiological state of the photosynthetic cell and triggers an extensive supramolecular reorganization of the photosynthetic membranes. The resulting picture of the photosynthetic apparatus is that of a highly flexible energy convertor that adapts to the ever-changing intracellular demand for ATP and/or reducing power.     

Snapshots of native pre-50S ribosomes reveal a biogenesis factor network and evolutionary specialization

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Analysis of sequence conservation at nucleotide resolution

One of the major goals of comparative genomics is to understand the evolutionary history of each nucleotide in the human genome sequence, and the degree to which it is under selective pressure. Ascertainment of selective constraint at nucleotide resolution is particularly important for predicting the functional significance of human genetic variation and for analyzing the sequence substructure of cis-regulatory sequences and other functional elements. Current methods for analysis of sequence conservation are focused on delineation of conserved regions comprising tens or even hundreds of consecutive nucleotides. We therefore developed a novel computational approach designed specifically for scoring evolutionary conservation at individual base-pair resolution. Our approach estimates the rate at which each nucleotide position is evolving, computes the probability of neutrality given this rate estimate, and summarizes the result in a Sequence CONservation Evaluation (SCONE) score. We computed SCONE scores in a continuous fashion across 1% of the human genome for which high-quality sequence information from up to 23 genomes are available. We show that SCONE scores are clearly correlated with the allele frequency of human polymorphisms in both coding and noncoding regions. We find that the majority of noncoding conserved nucleotides lie outside of longer conserved elements predicted by other conservation analyses, and are experiencing ongoing selection in modern humans as evident from the allele frequency spectrum of human polymorphism. We also applied SCONE to analyze the distribution of conserved nucleotides within functional regions. These regions are markedly enriched in individually conserved positions and short (<15 bp) conserved “chunks.” Our results collectively suggest that the majority of functionally important noncoding conserved positions are highly fragmented and reside outside of canonically defined long conserved noncoding sequences. A small subset of these fragmented positions may be identified with high confidence.     

Molecular dynamics simulations of RNA kissing-loop motifs reveal structural dynamics and formation of cation-binding pockets

Explicit solvent molecular dynamics (MD) simulations were carried out for three RNA kissing–loop complexes. The theoretical structure of two base pairs (2 bp) complex of H3 stem–loop of Moloney murine leukemia virus agrees with the NMR structure with modest violations of few NMR restraints comparable to violations present in the NMR structure. In contrast to the NMR structure, however, MD shows relaxed intermolecular G-C base pairs. The core region of the kissing complex forms a cation-binding pocket with highly negative electrostatic potential. The pocket shows nanosecond-scale breathing motions coupled with oscillations of the whole molecule. Additional simulations were carried out for 6 bp kissing complexes of the DIS HIV-1 subtypes A and B. The simulated structures agree well with the X-ray data. The subtype B forms a novel four-base stack of bulged-out adenines. Both 6 bp kissing complexes have extended cation-binding pockets in their central parts. While the pocket of subtype A interacts with two hexacoordinated Mg²⁺ ions and one sodium ion, pocket of subtype B is filled with a string of three delocalized Na⁺ ions with residency times of individual cations 1–2 ns. The 6 bp complexes show breathing motions of the cation-binding pockets and loop major grooves.     

Snapshots of the RNA editing machine in trypanosomes captured at different assembly stages in vivo

Mitochondrial pre‐messenger RNAs in kinetoplastid protozoa are substrates of uridylate‐specific RNA editing. RNA editing converts non‐functional pre‐mRNAs into translatable molecules and can generate protein diversity by alternative editing. Although several editing complexes have been described, their structure and relationship is unknown. Here, we report the isolation of functionally active RNA editing complexes by a multistep purification procedure. We show that the endogenous isolates contain two subpopulations of ～20S and ～35–40S and present the three‐dimensional structures of both complexes by electron microscopy. The ～35–40S complexes consist of a platform density packed against a semispherical element. The ～20S complexes are composed of two subdomains connected by an interface. The two particles are structurally related, and we show that RNA binding is a main determinant for the interconversion of the two complexes. The ～20S editosomes contain an RNA‐binding site, which binds gRNA, pre‐mRNA and gRNA/pre‐mRNA hybrid molecules with nanomolar affinity. Variability analysis indicates that subsets of complexes lack or possess additional domains, suggesting binding sites for components. Together, a picture of the RNA editing machinery is provided.     

Nature of eukaryotic proteins required for joining of 40S and 60S ribosomal subunits

A mixture of 40S and 60S subunits from salt-washed rabbit reticulocyte ribosomes fails to promote methionyl-puromycin synthesis under conditions in which an AUG-40S-Met-tRNA_i initiation complex, but not an 80S complex, is readily formed. This suggests that the inability of the system to form methionyl-puromycin is due to failure of the subunits to join. When $\text{Artemia salina}$ 60S subunits are substituted for their reticulocyte counterparts, the resulting hybrid system readily forms an 80S initiation complex and synthesizes methionyl-puromycin. Activity of the reticulocyte 60S subunits can be restored by factors IF-M2A and IF-M2B. This suggests that one or both of these factors may be 60S proteins, essential for subunit joining, that may be removed from ribosomes by salt washing procedures.     

Molecular dynamics simulations reveal insights into key structural elements of adenosine receptors

The crystal structure of the human A(2A) adenosine receptor, a member of the G protein-coupled receptor (GPCR) family, is used as a starting point for the structural characterization of the conformational equilibrium around the inactive conformation of the human A(2) (A(2A) and A(2B)) adenosine receptors (ARs). A homology model of the closely related A(2B)AR is reported, and the two receptors were simulated in their apo form through all-atom molecular dynamics (MD) simulations. Different conditions were additionally explored in the A(2A)AR, including the protonation state of crucial histidines or the presence of the cocrystallized ligand. Our simulations reveal the role of several conserved residues in the ARs in the conformational equilibrium of the receptors. The "ionic lock" absent in the crystal structure of the inactive A(2A)AR is rapidly formed in the two simulated receptors, and a complex network of interacting residues is presented that further stabilizes this structural element. Notably, the observed rotameric transition of Trp6.48 ("toggle switch"), which is thought to initiate the activation process in GPCRs, is accompanied by a concerted rotation of the conserved residue of the A(2)ARs, His6.52. This new conformation is further stabilized in the two receptors under study by a novel interaction network involving residues in transmembrane (TM) helices TM5 (Asn5.42) and TM3 (Gln3.37), which resemble the conformational changes recently observed in the agonist-bound structure of β-adrenoreceptors. Finally, the interaction between Glu1.39 and His7.43, a pair of conserved residues in the family of ARs, is found to be weaker than previously thought, and the role of this interaction in the structure and dynamics of the receptor is thoroughly examined. All these findings suggest that, despite the commonalities with other GPCRs, the conformational equilibrium of ARs is also modulated by specific residues of the family.     

Molecular dynamics simulations reveal structural coordination of Ffh-FtsY heterodimer toward GTPase activation

Two homologous GTPases (guanine-triphosphatases) in the signal recognition particle (SRP) and its receptor (SR) use their cumulative energy during GTP (guanine-triphosphate) hydrolysis to control the co-translational protein targeting process. Distinct from classical GTPases, which rely on external factors to hydrolyze GTP, SRP GTPases stimulate one another's activity in a self-sufficient manner upon SRP-SR complex association. Although both ground-state and putative transition-state GTP analogs have been used to recapitulate the state of GTPase activation, the underlying mechanism of the activated state still remains elusive. In particular, several residues that were placed in pending positions have been shown to be important to GTP hydrolysis in biochemical studies. Here, we examined the stability and dynamics of three interaction networks involving these residues and discovered that they contribute to the GTPase activation via well-tuned conformational changes. The crystallographically identified pending residues Ffh:R191/FtsY:R195 undergo extensive conformational rearrangements to form persisted interactions with FtsY:E284/Ffh:E274, explaining the biochemically observed defective effect of R191 mutant to the activation of both GTPases. In addition, the side chain of FtsY:R142, one of the most important catalytic residues, rotates to an extended conformation that could more efficiently maintain the electrostatic balance for GTP hydrolysis. Finally, the invariant residues Ffh:G190 and FtsY:G194, instead of the supposed auxiliary water molecules, are proposed to stabilize the nucleophilic waters during GTPase activation. In complementary to experimental observations, these findings suggest a more favorable interaction model for SRP GTPase activation and would thus benefit to our understanding of how SRP GTPases regulate the protein targeting pathway.     

FitEM2EM--tools for low resolution study of macromolecular assembly and dynamics

Studies of the structure and dynamics of macromolecular assemblies often involve comparison of low resolution models obtained using different techniques such as electron microscopy or atomic force microscopy. We present new computational tools for comparing (matching) and docking of low resolution structures, based on shape complementarity. The matched or docked objects are represented by three dimensional grids where the value of each grid point depends on its position with regard to the interior, surface or exterior of the object. The grids are correlated using fast Fourier transformations producing either matches of related objects or docking models depending on the details of the grid representations. The procedures incorporate thickening and smoothing of the surfaces of the objects which effectively compensates for differences in the resolution of the matched/docked objects, circumventing the need for resolution modification. The presented matching tool FitEM2EMin successfully fitted electron microscopy structures obtained at different resolutions, different conformers of the same structure and partial structures, ranking correct matches at the top in every case. The differences between the grid representations of the matched objects can be used to study conformation differences or to characterize the size and shape of substructures. The presented low-to-low docking tool FitEM2EMout ranked the expected models at the top.     

Electron microscopic study of eukaryotic 40S initiation complex in protein synthesis

This electron microscopic study demonstrates that formation of a functional eukaryotic 40S initiation complex is accompanied by conformational changes which obscure the characteristic structural features of the 40S ribosomal subunits and of the initiation factor eIF-3, the only macromolecular components of the complex individually resolvable by conventional high resolution electron microscopy. The complex, characterized by a sedimentation coefficient of 46S, appears as a globular particle with a diameter of about 280 A and several characteristic protrusions and incisions. Similar structures were obtained with [40S X eIF-3] initiation complexes formed by interaction of eIF-3 from rabbit reticulocytes with 40S ribosomal subunits from either A. salina cysts or mouse liver. Incubation of eIF-3 with prokaryotic 30S subunits from E. coli produced no [30S X eIF-3] structures. The binding of eIF-3 to 40S subunits is weak, and both the [40S X eIF-3] and the complete 40S initiation complexes have to be stabilized by glutaraldehyde fixation. The extensive conformational changes associated with the complex formation preclude direct electron microscopic localization of eIF-3, a globular protein approximately 100 A in diameter, in the initiation domain of the 40S subunit.     

Structure of a eukaryotic cytoplasmic pre‐40S ribosomal subunit

Final maturation of eukaryotic ribosomes occurs in the cytoplasm and requires the sequential removal of associated assembly factors and processing of the immature 20S pre‐RNA. Using cryo‐electron microscopy (cryo‐EM), we have determined the structure of a yeast cytoplasmic pre‐40S particle in complex with Enp1, Ltv1, Rio2, Tsr1, and Pno1 assembly factors poised to initiate final maturation. The structure reveals that the pre‐rRNA adopts a highly distorted conformation of its 3′ major and 3′ minor domains stabilized by the binding of the assembly factors. This observation is consistent with a mechanism that involves concerted release of the assembly factors orchestrated by the folding of the rRNA in the head of the pre‐40S subunit during the final stages of maturation. Our results provide a structural framework for the coordination of the final maturation events that drive a pre‐40S particle toward the mature form capable of engaging in translation.     

The nucleotide switch of tubulin and microtubule assembly: a polymerization-driven structural change

GTP-binding proteins from the tubulin family, including alphabeta-tubulin, gamma-tubulin, bacterial tubulin, and FtsZ, are key components of the cytoskeleton and play central roles in chromosome segregation and cell division. The nucleotide switch of alphabeta-tubulin is triggered by GTP hydrolysis and regulates microtubule assembly dynamics. The structural mechanism of the switch and how it modulates assembly are beginning to be understood. A conserved structural change between the active and inactive states, different from other GTPases, may be extracted from recent tubulin and FtsZ structures. From these and the biochemical properties of tubulin, the new concept emerges that, contrary to what was thought, unassembled tubulin-GTP is in the inactive, curved conformation as in tubulin-GDP rings, and it is driven into the straight microtubule conformation by the assembly contacts; binding of the GTP gamma-phosphate only lowers the free energy difference between the curved and straight forms.     

Micromanipulation studies of chromatin fibers in Xenopus egg extracts reveal ATP-dependent chromatin assembly dynamics

We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN). We observed force-induced disassembly and opening-closing fluctuations, indicating our experiments were in mechanochemical equilibrium. Roughly 50-nm (150-base pair) lengthening events dominated force-driven disassembly, suggesting that the assembled fibers are chiefly composed of nucleosomes. The ATP-depleted reaction was able to do mechanical work of 27 kcal/mol per 50 nm step, which provides an estimate of the free energy difference between core histone octamers on and off DNA. Addition of ATP led to highly dynamic behavior with time courses exhibiting processive runs of assembly and disassembly not observed in the ATP-depleted case. With ATP present, application of forces of 2 pN led to nearly complete fiber disassembly. Our study suggests that ATP hydrolysis plays a major role in nucleosome rearrangement and removal and that chromatin in vivo may be subject to highly dynamic assembly and disassembly processes that are modulated by DNA tension.     

Conformational flexibility and structural variability of SARS-CoV2 S protein

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