Structural and biochemical characterization of cauliflower mosaic virus reverse transcriptase.

Reverse transcriptases (RTs) are enzymes with DNA polymerase and RNase H activities. They convert single-stranded RNA into double-stranded DNA and are key enzymes for the replication of retroviruses and retroelements. Caulimoviridae is a major family of plant-infecting viruses. Caulimoviruses have a circular dsDNA genome that is replicated by reverse transcription, but in contrast to retroviruses, they lack integrase. Caulimoviruses are related to Ty3 retroelements. Ty3 RT has been extensively studied structurally and biochemically, but corresponding information for caulimoviral RTs is unavailable. In the present study, we report the first crystal structure of cauliflower mosaic virus (CaMV) RT in complex with a duplex made of RNA and DNA strands (RNA/DNA hybrid). CaMV RT forms a monomeric complex with the hybrid, unlike Ty3 RT, which does so as a dimer. Results of the RNA-dependent DNA polymerase and DNA-dependent DNA polymerase activity assays showed that individual CaMV RT molecules are able to perform full polymerase functions. However, our analyses showed that an additional CaMV RT molecule needs to transiently associate with a polymerase-competent RT molecule to execute RNase H cuts of the RNA strand. Collectively, our results provide details into the structure and function of CaMV RT and describe how the enzyme compares to other related RTs.

Toward an understanding of the conformational plasticity of S100A8 and S100A9 Ca2+-binding proteins.

S100A8 and S100A9 are small, human, Ca2+-binding proteins with multiple intracellular and extracellular functions in signaling, regulation, and defense. The two proteins are not detected as monomers but form various noncovalent homo- or hetero-oligomers related to specific activities in human physiology. Because of their significant roles in numerous medical conditions, there has been intense research on the conformational properties of various S100A8 and S100A9 proteoforms as essential targets of drug discovery. NMR or crystal structures are currently available only for mutated or truncated protein complexes, mainly with bound metal ions, that may well reflect the proteins' properties outside cells but not in other biological contexts in which they perform. Here, we used structural mass spectrometry methods combined with molecular dynamics simulations to compare the conformations of wildtype full-length S100A8 and S100A9 subunits in biologically relevant homo- and heterodimers and in higher oligomers formed in the presence of calcium or zinc ions. We provide, first, rationales for their functional response to changing environmental conditions, by elucidating differences between proteoforms in flexible protein regions that may provide the plasticity of the binding sites for the multiple targets, and second, the key factors contributing to the variable stability of the oligomers. The described methods and a systematic view of the conformational properties of S100A8 and S100A9 complexes provide a basis for further research to characterize and modulate their functions for basic science and therapies.

Quaternary organization of the human eEF1B complex reveals unique multi-GEF domain assembly.

Protein synthesis in eukaryotic cell is spatially and structurally compartmentalized that ensures high efficiency of this process. One of the distinctive features of higher eukaryotes is the existence of stable multi-protein complexes of aminoacyl-tRNA synthetases and translation elongation factors. Here, we report a quaternary organization of the human guanine-nucleotide exchange factor (GEF) complex, eEF1B, comprising α, ß and γ subunits that specifically associate into a heterotrimeric form eEF1B(αßγ)3. As both the eEF1Bα and eEF1Bß proteins have structurally conserved GEF domains, their total number within the complex is equal to six. Such, so far, unique structural assembly of the guanine-nucleotide exchange factors within a stable complex may be considered as a 'GEF hub' that ensures efficient maintenance of the translationally active GTP-bound conformation of eEF1A in higher eukaryotes.

Structures of Substrate Complexes of Foamy Viral Protease-Reverse Transcriptase.

Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA (dsDNA), a crucial process for the replication of retroviruses. Foamy viruses (FVs) possess a unique RT, which is a fusion with the protease (PR) domain. The mechanism of substrate binding by this enzyme has been unknown. Here, we report a crystal structure of monomeric full-length marmoset FV (MFV) PR-RT in complex with an RNA/DNA hybrid substrate. We also describe a structure of MFV PR-RT with an RNase H deletion in complex with a dsDNA substrate in which the enzyme forms an asymmetric homodimer. Cryo-electron microscopy reconstruction of the full-length MFV PR-RT-dsDNA complex confirmed the dimeric architecture. These findings represent the first structural description of nucleic acid binding by a foamy viral RT and demonstrate its ability to change its oligomeric state depending on the type of bound nucleic acid. IMPORTANCE Reverse transcriptases (RTs) are intriguing enzymes converting single-stranded RNA to dsDNA. Their activity is essential for retroviruses, which are divided into two subfamilies differing significantly in their life cycles: Orthoretrovirinae and Spumaretrovirinae. The latter family is much more ancient and comprises five genera. A unique feature of foamy viral RTs is that they contain N-terminal protease (PR) domains, which are not present in orthoretroviral enzymes. So far, no structural information for full-length foamy viral PR-RT interacting with nucleic substrates has been reported. Here, we present crystal and cryo-electron microscopy structures of marmoset foamy virus (MFV) PR-RT. These structures revealed the mode of binding of RNA/DNA and dsDNA substrates. Moreover, unexpectedly, the structures and biochemical data showed that foamy viral PR-RT can adopt both a monomeric configuration, which is observed in our structures in the presence of an RNA/DNA hybrid, and an asymmetric dimer arrangement, which we observed in the presence of dsDNA.

Vimentin S-glutathionylation at Cys328 inhibits filament elongation and induces severing of mature filaments in vitro.

Vimentin intermediate filaments are a significant component of the cytoskeleton in cells of mesenchymal origin. In vivo, filaments assemble and disassemble and thus participate in the dynamic processes of the cell. Post-translational modifications (PTMs) such as protein phosphorylation regulate the multiphasic association of vimentin from soluble complexes to insoluble filaments and the reverse processes. The thiol side chain of the single vimentin cysteine at position 328 (Cys328) is a direct target of oxidative modifications inside cells. Here, we used atomic force microscopy, electron microscopy and a novel hydrogen-deuterium exchange mass spectrometry (HDex-MS) procedure to investigate the structural consequences of S-nitrosylation and S-glutathionylation of Cys328 for in vitro oligomerisation of human vimentin. Neither modification affects the lateral association of tetramers to unit-length filaments (ULF). However, S-glutathionylation of Cys328 blocks the longitudinal assembly of ULF into extended filaments. S-nitrosylation of Cys328 does not hinder but slows down the elongation. Likewise, S-glutathionylation of preformed vimentin filaments causes their extensive fragmentation to smaller oligomeric species. Chemical reduction of the S-glutathionylated Cys328 thiols induces reassembly of the small fragments into extended filaments. In conclusion, our in vitro results suggest S-glutathionylation as a candidate PTM for an efficient molecular switch in the dynamic rearrangements of vimentin intermediate filaments, observed in vivo, in response to changes in cellular redox status. Finally, we demonstrate that HDex-MS is a powerful method for probing the kinetics of vimentin filament formation and filament disassembly induced by PTMs.

Crystal Structure and Directed Evolution of Specificity of NlaIV Restriction Endonuclease.

Specificity engineering is challenging and particularly difficult for enzymes that have the catalytic machinery and specificity determinants in close proximity. Restriction endonucleases have been used as a paradigm for protein engineering, but successful cases are rare. Here, we present the results of a directed evolution approach to the engineering of a dimeric, blunt end cutting restriction enzyme NlaIV (GGN/NCC). Based on the remote similarity to EcoRV endonuclease, regions for random mutagenesis and in vitro evolution were chosen. The obtained variants cleaved target sites with an up to 100-fold kcat/KM preference for AT or TA (GGW/WCC) over GC or CG (GGS/SCC) in the central dinucleotide step, compared to the only ~17-fold preference of the wild-type enzyme. To understand the basis of the increased specificity, we determined the crystal structure of NlaIV. Despite the presence of DNA in the crystallization mix, the enzyme crystallized in the free form. We therefore constructed a computational model of the NlaIV-DNA complex. According to the model, the mutagenesis of the regions that were in the proximity of DNA did not lead to the desired specificity change, which was instead conveyed in an indirect manner by substitutions in the more distant regions.

Enhanced oligomerization of full-length RAGE by synergy of the interaction of its domains.

The pattern recognition receptor RAGE (receptor for advanced glycation end-products) transmits proinflammatory signals in several inflammation-related pathological states, including vascular diseases, cancer, neurodegeneration and diabetes. Its oligomerization is believed to be important in signal transduction, but RAGE oligomeric structures and stoichiometries remain unclear. Different oligomerization modes have been proposed in studies involving different truncated versions of the extracellular parts of RAGE. Here, we provide basic characterization of the oligomerization patterns of full-length RAGE (including the transmembrane (TM) and cytosolic regions) and compare the results with oligomerization modes of its four truncated fragments. For this purpose, we used native mass spectrometry, analytical ultracentrifugation, and size-exclusion chromatography coupled with multi-angle light scattering. Our results confirm known oligomerization tendencies of separate domains and highlight the enhanced oligomerization properties of full-length RAGE. Mutational analyses within the GxxxG motif of the TM region show sensitivity of oligomeric distributions to the TM sequence. Using hydrogen-deuterium exchange, we mapped regions involved in TM-dependent RAGE oligomerization. Our data provide experimental evidence for the major role of the C2 and TM domains in oligomerization, underscoring synergy among different oligomerization contact regions along the RAGE sequence. These results also explain the variability of obtained oligomerization modes in RAGE fragments.

The protein-binding N-terminal domain of human translation elongation factor 1Bß possesses a dynamic α-helical structural organization.

Translation elongation factor 1Bß (eEF1Bß) is a metazoan-specific protein involved into the macromolecular eEF1B complex, containing also eEF1Bα and eEF1Bγ subunits. Both eEF1Bα and eEF1Bß ensure the guanine nucleotide exchange on eEF1A while eEF1Bγ is thought to have a structural role. The structures of the eEF1Bß catalytic C-terminal domain and neighboring central acidic region are known while the structure of the protein-binding N-terminal domain remains unidentified which prevents clear understanding of architecture of the eEF1B complex. Here we show that the N-terminal domain comprising initial 77 amino acids of eEF1Bß, eEF1Bß(1-77), is a monomer in solution with increased hydrodynamic volume. This domain binds eEF1Bγ in equimolar ratio. The CD spectra reveal that the secondary structure of eEF1Bß(1-77) consists predominantly of α-helices and a portion of disordered region. Very rapid hydrogen/deuterium exchange for all eEF1Bß(1-77) peptides favors a flexible tertiary organization of eEF1Bß(1-77). Computational modeling of eEF1Bß(1-77) suggests several conformation states each composed of three α-helices connected by flexible linkers. Altogether, the data imply that the protein-binding domain of eEF1Bß shows flexible spatial organization which may be needed for interaction with eEF1Bγ or other protein partners.

The Y. bercovieri Anbu crystal structure sheds light on the evolution of highly (pseudo)symmetric multimers.

Ancestral ß-subunit (Anbu) is homologous to HslV and 20S proteasomes. Based on its phylogenetic distribution and sequence clustering, Anbu has been proposed as the "ancestral" form of proteasomes. Here, we report biochemical data, small-angle X-ray scattering results, negative-stain electron microscopy micrographs and a crystal structure of the Anbu particle from Yersinia bercovieri (YbAnbu). All data are consistent with YbAnbu forming defined 12-14 subunit multimers that differ in shape from both HslV and 20S proteasomes. The crystal structure reveals that YbAnbu subunits form tight dimers, held together in part by the Anbu specific C-terminal helices. These dimers ("protomers") further assemble into a low-rise left-handed staircase. The lock-washer shape of YbAnbu is consistent with the presence of defined multimers, X-ray diffraction data in solution and negative-stain electron microscopy images. The presented structure suggests a possible evolutionary pathway from helical filaments to highly symmetric or pseudosymmetric multimer structures. YbAnbu subunits have the Ntn-hydrolase fold, a putative S1 pocket and conserved candidate catalytic residues Thr1, Asp17 and Lys32(33). Nevertheless, we did not detect any YbAnbu peptidase or amidase activity. However, we could document orthophosphate production from ATP catalyzed by the ATP-grasp protein encoded in the Y. bercovieri Anbu operon.

A non-catalytic N-terminal domain negatively influences the nucleotide exchange activity of translation elongation factor 1Bα.

Eukaryotic translation elongation factor 1Bα (eEF1Bα) is a functional homolog of the bacterial factor EF-Ts, and is a component of the macromolecular eEF1B complex. eEF1Bα functions as a catalyst of guanine nucleotide exchange on translation elongation factor 1A (eEF1A). The C-terminal domain of eEF1Bα is necessary and sufficient for its catalytic activity, whereas the N-terminal domain interacts with eukaryotic translation elongation factor 1Bγ (eEF1Bγ) to form a tight complex. However, eEF1Bγ has been shown to enhance the catalytic activity of eEF1Bα attributed to the C-terminal domain of eEF1Bα. This suggests that the N-terminal domain of eEF1Bα may in some way influence the guanine nucleotide exchange process. We have shown that full-length recombinant eEF1Bα and its truncated forms are non-globular proteins with elongated shapes. Truncation of the N-terminal domain of eEF1Bα, which is dispensable for catalytic activity, resulted in acceleration of the rate of guanine nucleotide exchange on eEF1A compared to full-length eEF1Bα. A similar effect on the catalytic activity of eEF1Bα was observed after its interaction with eEF1Bγ. We suggest that the non-catalytic N-terminal domain of eEF1Bα may interfere with eEF1A binding to the C-terminal catalytic domain, resulting in a decrease in the overall rate of the guanine nucleotide exchange reaction. Formation of a tight complex between the eEF1Bγ and eEF1Bα N-terminal domains abolishes this inhibitory effect.

Structural and Mechanistic Analysis of the Slx1-Slx4 Endonuclease.

The SLX1-SLX4 endonuclease required for homologous recombination and DNA repair in eukaryotic cells cleaves a variety of branched DNA structures. The nuclease subunit SLX1 is activated by association with a scaffolding protein SLX4. At the present time, little is known about the structure of SLX1-SLX4 or its mechanism of action. Here, we report the structural insights into SLX1-SLX4 by detailing the crystal structure of Candida glabrata (Cg) Slx1 alone and in combination with the C-terminal region of Slx4. The structure of Slx1 reveals a compact arrangement of the GIY-YIG nuclease and RING domains, which is reinforced by a long α helix. Slx1 forms a stable homodimer that blocks its active site. Slx1-Slx4 interaction is mutually exclusive with Slx1 homodimerization, suggesting a mechanism for Slx1 activation by Slx4.

2-Thiouracil deprived of thiocarbonyl function preferentially base pairs with guanine rather than adenine in RNA and DNA duplexes.

2-Thiouracil-containing nucleosides are essential modified units of natural and synthetic nucleic acids. In particular, the 5-substituted-2-thiouridines (S2Us) present in tRNA play an important role in tuning the translation process through codon-anticodon interactions. The enhanced thermodynamic stability of S2U-containing RNA duplexes and the preferred S2U-A versus S2U-G base pairing are appreciated characteristics of S2U-modified molecular probes. Recently, we have demonstrated that 2-thiouridine (alone or within an RNA chain) is predominantly transformed under oxidative stress conditions to 4-pyrimidinone riboside (H2U) and not to uridine. Due to the important biological functions and various biotechnological applications for sulfur-containing nucleic acids, we compared the thermodynamic stabilities of duplexes containing desulfured products with those of 2-thiouracil-modified RNA and DNA duplexes. Differential scanning calorimetry experiments and theoretical calculations demonstrate that upon 2-thiouracil desulfuration to 4-pyrimidinone, the preferred base pairing of S2U with adenosine is lost, with preferred base pairing with guanosine observed instead. Therefore, biological processes and in vitro assays in which oxidative desulfuration of 2-thiouracil-containing components occurs may be altered. Moreover, we propose that the H2U-G base pair is a suitable model for investigation of the preferred recognition of 3'-G-ending versus A-ending codons by tRNA wobble nucleosides, which may adopt a 4-pyrimidinone-type structural motif.

Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress.

CacyBP/SIP [calcyclin-binding protein/Siah-1 [seven in absentia homolog 1 (Siah E3 ubiquitin protein ligase 1)] interacting protein] is a multifunctional protein whose activity includes acting as an ERK1/2 phosphatase. We analyzed dimerization of mouse CacyBP/SIP in vitro and in mouse neuroblastoma cell line (NB2a) cells, as well as the structure of a full-length protein. Moreover, we searched for the CacyBP/SIP domain important for dimerization and dephosphorylation of ERK2, and we analyzed the role of dimerization in ERK1/2 signaling in NB2a cells. Cell-based assays showed that CacyBP/SIP forms a homodimer in NB2a cell lysate, and biophysical methods demonstrated that CacyBP/SIP forms a stable dimer in vitro. Data obtained using small-angle X-ray scattering supported a model in which CacyBP/SIP occupies an anti-parallel orientation mediated by the N-terminal dimerization domain. Site-directed mutagenesis established that the N-terminal domain is indispensable for full phosphatase activity of CacyBP/SIP. We also demonstrated that the oligomerization state of CacyBP/SIP as well as the level of post-translational modifications and subcellular distribution of CacyBP/SIP change after activation of the ERK1/2 pathway in NB2a cells due to oxidative stress. Together, our results suggest that dimerization is important for controlling phosphatase activity of CacyBP/SIP and for regulating the ERK1/2 signaling pathway.

Patterns of structural dynamics in RACK1 protein retained throughout evolution: a hydrogen-deuterium exchange study of three orthologs.

RACK1 is a member of the WD repeat family of proteins and is involved in multiple fundamental cellular processes. An intriguing feature of RACK1 is its ability to interact with at least 80 different protein partners. Thus, the structural features enabling such interactomic flexibility are of great interest. Several previous studies of the crystal structures of RACK1 orthologs described its detailed architecture and confirmed predictions that RACK1 adopts a seven-bladed ß-propeller fold. However, this did not explain its ability to bind to multiple partners. We performed hydrogen-deuterium (H-D) exchange mass spectrometry on three orthologs of RACK1 (human, yeast, and plant) to obtain insights into the dynamic properties of RACK1 in solution. All three variants retained similar patterns of deuterium uptake, with some pronounced differences that can be attributed to RACK1's divergent biological functions. In all cases, the most rigid structural elements were confined to B-C turns and, to some extent, strands B and C, while the remaining regions retained much flexibility. We also compared the average rate constants for H-D exchange in different regions of RACK1 and found that amide protons in some regions exchanged at least 1000-fold faster than in others. We conclude that its evolutionarily retained structural architecture might have allowed RACK1 to accommodate multiple molecular partners. This was exemplified by our additional analysis of yeast RACK1 dimer, which showed stabilization, as well as destabilization, of several interface regions upon dimer formation.

Ty3 reverse transcriptase complexed with an RNA-DNA hybrid shows structural and functional asymmetry.

Retrotransposons are a class of mobile genetic elements that replicate by converting their single-stranded RNA intermediate to double-stranded DNA through the combined DNA polymerase and ribonuclease H (RNase H) activities of the element-encoded reverse transcriptase (RT). Although a wealth of structural information is available for lentiviral and gammaretroviral RTs, equivalent studies on counterpart enzymes of long terminal repeat (LTR)-containing retrotransposons, from which they are evolutionarily derived, is lacking. In this study, we report the first crystal structure of a complex of RT from the Saccharomyces cerevisiae LTR retrotransposon Ty3 in the presence of its polypurine tract-containing RNA-DNA hybrid. In contrast to its retroviral counterparts, Ty3 RT adopts an asymmetric homodimeric architecture whose assembly is substrate dependent. Moreover, our structure and biochemical data suggest that the RNase H and DNA polymerase activities are contributed by individual subunits of the homodimer.

NMR structural studies of the first catalytic half-domain of ubiquitin activating enzyme.

We report a high resolution NMR structure and (15)N relaxation studies of the first catalytic cysteine half-domain (FCCH) of the mouse ubiquitin-activating enzyme E1, together with interaction studies of FCCH and the other catalytic E1 subdomain - SCCH (second catalytic cysteine half-domain). In solution, mouse FCCH forms a well-defined six-stranded antiparallel ß-barrel structure, a common fold for many proteins with a variety of cellular functions. (15)N relaxation data reveal FCCH complex backbone dynamics and indicate which residues experience slow intramolecular motions. Some of these residues make contacts with the polar face of ubiquitin in the co-crystal structure of yeast E1 and ubiquitin. However, the titration of FCCH with ubiquitin does not show any visible chemical shift changes in the 2D (1)H/(15)N HSQC spectra of the FCCH. The 2D (1)H/(15)N HSQC experiments performed both for each catalytic half-domain individually and for their equimolar mixture in the milimolar concentration range display no detectable chemical shift perturbation, suggesting a lack of interaction between the two subdomains unless they are covalently linked via the adenylation domain.

Central base pair flipping and discrimination by PspGI.

PspGI is a representative of a group of restriction endonucleases that recognize a pentameric sequence related to CCNGG. Unlike the previously investigated Ecl18kI, which does not have any specificity for the central base pair, PspGI prefers A/T over G/C in its target site. Here, we present a structure of PspGI with target DNA at 1.7 A resolution. In this structure, the bases at the center of the recognition sequence are extruded from the DNA and flipped into pockets of PspGI. The flipped thymine is in the usual anti conformation, but the flipped adenine takes the normally unfavorable syn conformation. The results of this and the accompanying manuscript attribute the preference for A/T pairs over G/C pairs in the flipping position to the intrinsically lower penalty for flipping A/T pairs and to selection of the PspGI pockets against guanine and cytosine. Our data show that flipping can contribute to the discrimination between normal bases. This adds a new role to base flipping in addition to its well-known function in base modification and DNA damage repair.

How PspGI, catalytic domain of EcoRII and Ecl18kI acquire specificities for different DNA targets.

Restriction endonucleases Ecl18kI and PspGI/catalytic domain of EcoRII recognize CCNGG and CCWGG sequences (W stands for A or T), respectively. The enzymes are structurally similar, interact identically with the palindromic CC:GG parts of their recognition sequences and flip the nucleotides at their centers. Specificity for the central nucleotides could be influenced by the strength/stability of the base pair to be disrupted and/or by direct interactions of the enzymes with the flipped bases. Here, we address the importance of these contributions. We demonstrate that wt Ecl18kI cleaves oligoduplexes containing canonical, mismatched and abasic sites in the central position of its target sequence CCNGG with equal efficiencies. In contrast, substitutions in the binding pocket for the extrahelical base alter the Ecl18kI preference for the target site: the W61Y mutant prefers only certain mismatched substrates, and the W61A variant cuts exclusively at abasic sites, suggesting that pocket interactions play a major role in base discrimination. PspGI and catalytic domain of EcoRII probe the stability of the central base pair and the identity of the flipped bases in the pockets. This 'double check' mechanism explains their extraordinary specificity for an A/T pair in the flipping position.

Thermodynamic studies of interactions of calf spleen PNP with acyclic phosphonate inhibitors.

The Gibbs binding energy and entropy/enthalpy contributions to the interaction of calf spleen purine nucleoside phosphorylase (PNP) with the novel multisubstrate analogue DFPP-DG, as well as with DFPP-G and (S)-PMP-DAP were determined by fluorescence and calorimetric studies. Results were compared with findings for guanine - a natural reaction product and inhibitor.

Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence.

Many DNA modification and repair enzymes require access to DNA bases and therefore flip nucleotides. Restriction endonucleases (REases) hydrolyze the phosphodiester backbone within or in the vicinity of the target recognition site and do not require base extrusion for the sequence readout and catalysis. Therefore, the observation of extrahelical nucleotides in a co-crystal of REase Ecl18kI with the cognate sequence, CCNGG, was unexpected. It turned out that Ecl18kI reads directly only the CCGG sequence and skips the unspecified N nucleotides, flipping them out from the helix. Sequence and structure conservation predict nucleotide flipping also for the complexes of PspGI and EcoRII with their target DNAs (/CCWGG), but data in solution are limited and indirect. Here, we demonstrate that Ecl18kI, the C-terminal domain of EcoRII (EcoRII-C) and PspGI enhance the fluorescence of 2-aminopurines (2-AP) placed at the centers of their recognition sequences. The fluorescence increase is largest for PspGI, intermediate for EcoRII-C and smallest for Ecl18kI, probably reflecting the differences in the hydrophobicity of the binding pockets within the protein. Omitting divalent metal cations and mutation of the binding pocket tryptophan to alanine strongly increase the 2-AP signal in the Ecl18kI-DNA complex. Together, our data provide the first direct evidence that Ecl18kI, EcoRII-C and PspGI flip nucleotides in solution.