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bioRxiv2017

Conformational locking of Ufm1 upon binding to the Ufm1-interacting sequence of Uba5

Ubiquitin fold modifier 1 (Ufm1) is a ubiquitin-like protein (UBL) found in eukaryotic organisms which plays a crucial role in ER stress management and signal transduction. The crystal structure of UFM1 and its E1 (Uba5) in complex shows that Ufm1 binds to the adenylation domain of UBA5 and interacts with a separate Ufm1-interacting sequence (UIS) in the C-terminus of UBA5. The UIS interacts with Ufm1 on the opposite side of Ufm1 protein from the adenylation domain of Uba5 and the reason for this second interaction site is unclear. We analyzed Ufm1 bound to the UIS sequence through molecular dynamics simulations in order to identify additional functions for this interaction. We found that the residues in the adenylation interaction site of Ufm1 have less movement when the UIS peptide was bound to Ufm1 and formed a structure that aligns well with Ufm1 bound to the Uba5 adenylation domain. We further identified an amino acid that connects the UIS to the adenylation domain interacting site. Mutation of this amino acid decreases charging activity and shifts the Ufm1 conformation population toward the unlocked configuration even in the presence of the UIS peptide. These data suggest a role for the Uba5 UIS in stimulating activation of Ufm1.

biochemistry↗

bioRxiv2017

A Careful Consideration of the Influence of Structure, Partial charges and Basis Sets on Collision Cross Sections of Monosaccharides when Comparing Values from DFT Calculated Conformers to those Obtained Experimentally

Molecular modelling is routinely employed to assign 3D structures to collision cross sections (CCSs) derived from ion mobility mass spectrometry experiments (IM-MS). The assignment of model structures to the experimental CCSs remains an ambiguous task, where one of several methods may be used to obtain a CCS from a given set of coordinates. The most reliable of the commonly used techniques, the Trajectory Method, starts with atomic coordinates which can be accompanied by partial atomic charges, obtained using ab initio methods. Here, we use lithiated - and {beta}-glucose ions as exemplar molecules to detect the effect conformational modification and changes to the partial charge distribution have on computed collision cross sections. Six popular charge schemes (Mulliken, APT, CHelpG, MK, HLY and NPA) were examined in combination with three functionals (Hartree-Fock, B3LYP and M05) and five basis sets (STO-3G, 3-21G, 6-31G, 6-31+G and 6-31G*) on twenty unique structures. Our findings indicate that molecular conformation makes a significant contribution to fluctuations of partial charges in Electrostatic Potential (ESP) and Mulliken charge scheme; Partial charges derived using Natural Population Analysis (NPA) and ESP methods are largely independent of functional and basis set selection; and both selection of the charge scheme and functional/basis set combination play a large role in the resultant CCS, often causing few percent fluctuations in the computed values.

biochemistry↗

bioRxiv2017

Monomerization of Far-Red Fluorescent Proteins

Anthozoa class red fluorescent proteins (RFPs) are frequently used as biological markers, with far-red emitting variants ({lambda}em ~ 600 - 900 nm) sought for whole animal imaging because biological tissues are permeable to light in this range. A barrier to the use of naturally occurring RFP variants as molecular markers is that all are tetrameric, which is not ideal for cell biological applications. Efforts to engineer monomeric RFPs have usually produced dimmer and blue-shifted variants, as the chromophore is sensitive to small structural perturbations. In fact, despite much effort, only four native RFPs have been successfully monomerized, leaving the vast majority of RFP biodiversity untapped in biomarker development. Here we report the generation of monomeric variants of HcRed and mCardinal, both far-red dimers, and describe a comprehensive methodology for the rapid monomerization of novel red-shifted oligomeric RFPs. Among the resultant variants, is mKelly1 (emission maximum: {lambda}em = 656 nm), which along with the recently reported mGarnet2 (1), forms a new class of bright, monomeric, far-red FPs.

biochemistry↗

bioRxiv2017

Picky: a simple method designer for targeted proteomics

Targeted proteomic approaches like selected reaction monitoring (SRM) and parallel reaction monitoring (PRM) are increasingly popular because they enable sensitive and rapid analysis of a preselected set of proteins1-3. However, developing targeted assays is tedious and requires the selection, synthesis and mass spectrometric analysis of candidate peptides before the actual samples can be analyzed. The SRMatlas and ProteomeTools projects recently published fragmentation spectra of synthetic peptides covering the entire human proteome4,5. These datasets provide very valuable resources. However, extracting the relevant data for selected proteins of interest is not straightforward. For example, developing scheduled acquisition methods (i.e. analyzing specific peptides in defined elution time windows) is complicated and requires adjustments to specific chromatographic conditions employed. Moreover, the number of peptide candidates to be targeted in parallel often exceeds the analytical abilities of the mass spectrometer. In this case, the key question is which peptides can be omitted without losing too much information. Ideally, a method design tool would automatically select the most informative peptides in each retention time window. Until now, none of the available tools automatically generates such optimized scheduled SRM and PRM methods (Figure S1).\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC=\"FIGDIR/small/163154_figS1.gif\" ALT=\"Figure 1\">\nView larger version (13K):\norg.highwire.dtl.DTLVardef@828org.highwire.dtl.DTLVardef@17a73aorg.highwire.dtl.DTLVardef@e40740org.highwire.dtl.DTLVardef@9816e7_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. S1:C_FLOATNO Comparison between different available SRM or PRM method generators.\n\nC_FIG

biochemistry↗

bioRxiv2017

The WYL domain of the Thermotoga elfii PIF1 helicase is an accessory single-stranded DNA binding module

PIF1 family helicases are conserved from bacteria to man. With the exception of the well-studied yeast PIF1 helicases (e.g., ScPif1 and ScRrm3), however, very little is known about how these enzymes help maintain genome stability. Indeed, we lack a basic understanding of the protein domains found N- and C-terminal to the characteristic central PIF1 helicase domain in these proteins. Here, using chimeric constructs, we show that the ScPif1 and ScRrm3 helicase domains are interchangeable and that the N-terminus of ScRrm3 is important for its function in vivo. This suggests that PIF1 family helicases evolved functional modules fused to a generic motor domain. To investigate this hypothesis, we characterized the biochemical activities of the PIF1 helicase from the thermophilic bacterium Thermotoga elfii (TePif1), which contains a C-terminal WYL domain of unknown function. Like helicases from other thermophiles, recombinant TePif1 was easily prepared, thermostable in vitro, and displayed activities similar to its eukaryotic homologs. We also found that the WYL domain was necessary for high-affinity single-stranded DNA (ssDNA) binding and affected both ATPase and helicase activities. Deleting the WYL domain from TePif1 or mutating conserved residues in the predicted ssDNA binding site uncoupled ATPase activity and DNA unwinding, leading to higher rates of ATP hydrolysis but less efficient DNA helicase activity. Our findings suggest that the domains of unknown function found in eukaryotic PIF1 helicases may also confer functional specificity and additional activities to these enzymes, which should be investigated in future work.

biochemistry↗

bioRxiv2017

Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes

Next-generation sequencing has engendered an expanding suite of functional assays that can test sequence-function relationships at unprecedented scales in pooled formats (multiplex). Such assays are currently constrained by the short length of oligonucleotide (oligo) pools, which limit potential applications. Here we report a simple, low-cost, and scalable method called DropSynth that assembles gene libraries from oligo pools for use in multiplexed functional assays. DropSynth utilizes a library of barcoded beads to isolate and concentrate oligos needed for a genes synthesis in a pooled format. These bead-bound oligos are then emulsified, processed, and assembled into genes within the emulsion droplets. We synthesized ~1000 phylogenetically diverse orthologs of phosphopantetheine adenylyltransferase (PPAT) and tested their fitness in a multiplexed functional assay. While the majority of orthologs complement, those that do not are broadly distributed across the phylogenetic tree. Synthetic errors in our assemblies allow us to explore local landscapes around the designed orthologs revealing constrained mutations for complementing orthologs as well as gain-of-function mutations for low-fitness orthologs. This broad mutational scanning approach is complementary to deep mutational scanning and helps us understand proteins by probing evolutionarily divergent sequences that share function.

biochemistry↗

bioRxiv2017

Interferon Regulatory Factor 5 (IRF5) Interacts with the Translation Initiation Complex and Promotes mRNA Translation During the Integrated Stress Response to Amino Acid Deprivation

Interferon Regulatory Factor 5 (IRF5) plays an important role in limiting pathogenic infection and tumor development. Host protection by IRF5 can occur through a variety of mechanisms including production of type I interferon and cytokines as well as the regulation of cell survival, growth, proliferation, and differentiation. While modulation of these cellular processes is attributed to IRF5 transcription factor function in the nucleus, emerging evidence suggests that IRF5 may also retain non-transcriptional regulatory properties within the cytoplasmic compartment. Consistent with this notion, we report the ability of IRF5 to control gene expression at the level of mRNA translation. Our findings demonstrate that IRF5 interacts with the translation initiation complex in the absence of the m7GTP cap-binding protein, eIF4E. We observed that under nutrient deprivation-induced cell stress, IRF5 promoted mRNA translation of the master integrated stress response (ISR) regulator, Activating Transcription Factor 4 (ATF4). Enhanced ATF4 protein expression correlated with increased levels of downstream target genes including CHOP and GADD34 and was associated with amplification of eIF2 de-phosphorylation and translational de-repression under stress. The novel mechanism we describe broadens our understanding of how IRF5 regulates gene expression and may govern diverse cellular processes in the absence of stimuli that trigger IRF5 nuclear translocation.

biochemistry↗

bioRxiv2017

Transition metal binding selectivity in proteins and its correlation with the phylogenomic classification of the cation diffusion facilitator protein family

Divalent d-block metal cations (DDMCs), such as Fe, Zn and Mn, participate in many biological processes. Understanding how specific DDMCs are transported to and within the cell and what controls their binding selectivity to different proteins is crucial for defining the mechanisms of metalloproteins. To better understand such processes, we scanned the RCSB Protein Data Bank, performed a de novo structural-based comprehensive analysis of seven DDMCs and found their amino acid binding and coordination geometry propensities. We then utilized these results to characterize the correlation between metal selectivity, specific binding site composition and phylogenetic classification of the cation diffusion facilitator (CDF) protein family, a family of DDMC transporters found throughout evolution and sharing a conserved structure, yet with different members displaying distinct metal selectivity. Our analysis shows that DDMCs differ, at times significantly, in terms of their binding propensities, and that in each CDF clade, the metal selectivity-related binding site has a unique and conserved sequence signature. However, only limited correlation exists between the composition of the DDMC binding site in each clade and the metal selectivity shown by its proteins.

biochemistry↗

bioRxiv2017

Juxtacap Nucleotide Sequence Modulates eIF4E Binding and Translation

mRNA translation is an energetically costly activity required for almost all biological processes. The multiprotein complex eIF4F, which bridges the 5 cap and the polyA tail through eIF4E and eIF4G, respectively, is necessary for efficient translation initiation of most mRNAs and is an important target of translational control. Previous work suggests that cap-proximal nucleotides can modulate eIF4E binding to mRNAs, but the effect of specific cap-proximal nucleotide sequences on eIF4E recruitment and the ultimate consequences for translation remain unknown. Using RNA Bind-n-Seq on a model 5 UTR, we systematically identify eIF4E-intrinsic cap-proximal nucleotide binding preferences. mRNAs with highly-bound motifs are translated well in a cell-free system, whereas those with low-eIF4E-binding motifs are not. However, eIF4E juxtacap motif preferences do not dictate the ribosome occupancy of endogenous mRNAs in cells, suggesting that the effect of juxtacap sequence on eIF4E binding and translation is mRNA-context-dependent. Accordingly, a single downstream point mutation that disrupts a predicted base pair with a preferred juxtacap nucleotide increases translation. The juxtacap sequence is a previously unappreciated determinant of eIF4E recruitment to mRNAs, and we propose that differences in mRNA 5 end accessibility defined by the juxtacap sequence are important for establishing translational efficiency.

biochemistry↗

bioRxiv2017

Dynamical origins of heat capacity changes in enzyme catalysed reactions

Heat capacity changes are emerging as essential for explaining the temperature dependence of enzyme-catalysed reaction rates. This has important implications for enzyme kinetics, thermoadaptation and evolution, but the physical basis of these heat capacity changes is unknown. Here we show by a combination of experiment and simulation, for two quite distinct enzymes (dimeric ketosteroid isomerase and monomeric alpha-glucosidase), that the activation heat capacity change for the catalysed reaction can be predicted through atomistic molecular dynamics simulations. The simulations reveal subtle and surprising underlying dynamical changes: tightening of loops around the active site is observed as expected, but crucially, changes in energetic fluctuations are evident across the whole enzyme including important contributions from oligomeric neighbours and domains distal to the active site. This has general implications for understanding enzyme catalysis and demonstrating a direct connection between functionally important microscopic dynamics and macroscopically measurable quantities.

biochemistry↗

bioRxiv2017

Prodomain-Growth Factor Swapping in the Structure of pro-TGF-β1

Transforming growth factor (TGF)-{beta} is synthesized as a proprotein that dimerizes in the endoplasmic reticulum. After processing in the Golgi to cleave the N-terminal prodomain from the C-terminal growth factor (GF) domain in each monomer, pro-TGF-{beta} is secreted and stored in latent complexes. It is unclear which prodomain and GF monomer are linked prior to proprotein convertase (PC) cleavage, and how much conformational change occurs following cleavage. We have determined a structure of pro-TGF-{beta}1 with the PC cleavage site mutated, to mimic the structure of the TGF-{beta}1 proprotein. Our structure demonstrates that the prodomain arm domain in one monomer is linked to the GF that interacts with the arm domain in the other monomer in the dimeric structure, i.e., the prodomain arm domain and GF domain in each monomer are swapped. Swapping has important implications for the mechanism of biosynthesis in the TGF-{beta} family and is relevant to the mechanism for preferential formation of heterodimers over homodimers for some members of the TGF-{beta} family. Our structure also provides comparisons between independent TGF-{beta}1 crystal structures and between human and porcine pro-TGF-{beta}1.

biochemistry↗

bioRxiv2017

Structure of the deactive state of mammalian respiratory complex I

Complex I (NADH:ubiquinone oxidoreductase) is central to energy metabolism in mammalian mitochondria. It couples NADH oxidation by ubiquinone to proton transport across the energy-conserving inner membrane, catalyzing respiration and driving ATP synthesis. In the absence of substrates, active complex I gradually enters a pronounced resting or deactive state. The active-deactive transition occurs during ischemia and is crucial for controlling how respiration recovers upon reperfusion. Here, we set a highly-active preparation of Bos taurus complex I into the biochemically-defined deactive state, and used single-particle electron cryomicroscopy to determine its structure to 4.1 [A] resolution. The deactive state arises when critical structural elements that form the ubiquinone-binding site become disordered, and we propose reactivation is induced when substrate binding templates their reordering. Our structure both rationalizes biochemical data on the deactive state, and offers new insights into its physiological and cellular roles.

biochemistry↗

bioRxiv2017

Regulatory coiled-coil domains promote head-to-head assemblies of AAA+ chaperones essential for tunable activity control

Ring-forming AAA+ chaperones exert ATP-fueled substrate unfolding by threading through a central pore. This activity is potentially harmful requiring mechanisms for tight repression and substrate-specific activation. The AAA+ chaperone ClpC with the peptidase ClpP forms a bacterial protease essential to virulence and stress resistance. The adaptor MecA activates ClpC by targeting substrates and stimulating ClpC ATPase activity. We show how ClpC is repressed in its ground state by determining ClpC cryo-EM structures with and without MecA. ClpC forms large two-helical assemblies that associate via head-to-head contacts between coiled-coil middle domains (MDs). MecA converts this resting state to an active planar ring structure by binding to MD interaction sites. Loss of ClpC repression in MD mutants causes constitutive activation and severe cellular toxicity. These findings unravel an unexpected regulatory concept executed by coiled-coil MDs to tightly control AAA+ chaperone activity.

biochemistry↗

bioRxiv2017

Pollen tube growth and guidance: Occam’s razor sharpened on a molecular AGP Rosetta Stone

Occams Razor suggests a new model of pollen tube tip growth based on a novel Hechtian oscillator that integrates: (1) a periplasmic AGP-Ca2+ calcium capacitor with tip-localised arabinogalactan glycoproteins (AGPs); (2) tip-focussed cytosolic Ca2+oscillations; (3) Hechtian strands evidence of adhesion between the plasma membrane and the cell wall of the growing tip. Thus Hechtian adhesion, as a piconewton force transducer, couples the internal stress of a rapidly growing wall to the plasma membrane. Such Hechtian transduction via stretch-activated Ca2+ channels and H+-ATPase proton efflux dissociating periplasmic AGP-Ca2+, creates a Ca2+ influx that activates exocytosis of wall precursors. In effect a highly simplified primary cell wall regulates its own synthesis and a Hechtian growth oscillator regulates overall tip growth. By analogy with the Rosetta Stone that translates trilingual inscriptions as a single identical proclamation, the Hechtian Hypothesis translates classical AGPs and their roles as a Ca2+ capacitor, pollen tube guide and wall plasticiser into a simple but widely applicable model of tip growth. Even wider ramifications of the Hechtian oscillator may implicate AGPs in osmosensing or gravisensing and other tropisms, leading us yet further towards the Holy Grail of plant growth.

biochemistry↗

bioRxiv2017

Architecture of TAF11/TAF13/TBP complex suggests novel regulatory state in General Transcription Factor TFIID function

General transcription factor TFIID is a key component of RNA polymerase II transcription initiation. Human TFIID is a megadalton-sized complex comprising TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). TBP binds to core promoter DNA, recognizing the TATA-box. We identified a ternary complex formed by TBP and the histone fold (HF) domain-containing TFIID subunits TAF11 and TAF13. We demonstrate that TAF11/TAF13 competes for TBP binding with TATA-box DNA, and also with the N-terminal domain of TAF1 previously implicated in TATA-box mimicry. In an integrative approach combining crystal coordinates, biochemical analyses and data from cross-linking mass-spectrometry (CLMS), we determine the architecture of the TAF11/TAF13/TBP complex, revealing TAF11/TAF13 interaction with the DNA binding surface of TBP. We identify a highly conserved C-terminal TBP-binding domain (CTID) in TAF13 which is essential for supporting cell growth. Our results thus have implications for cellular TFIID assembly and suggest a novel regulatory state for TFIID function.

biochemistry↗

bioRxiv2017

Structure of the triose-phosphate/phosphate translocator reveals the basis of substrate specificity

The triose-phosphate/phosphate translocator (TPT) catalyzes the strict 1:1 exchange of triose phosphate, 3-phosphoglycerate and inorganic phosphate across the chloroplast envelope, and plays crucial roles in photosynthesis. Despite rigorous studies for more than 40 years, the molecular mechanism of TPT is poorly understood due to the lack of structural information. Here we report crystal structures of TPT bound to two different substrates, 3-phosphoglycerate and inorganic phosphate, in occluded conformations. The structures reveal that TPT adopts a 10-transmembrane drug/metabolite transporter fold. Both substrates are bound within the same central pocket, where conserved lysine, arginine, and tyrosine residues recognize the shared phosphate group. A structural comparison with the outward-open conformation of the bacterial drug/metabolite transporter suggests a rocking-type motion of helix bundles, and molecular dynamics simulations support a model in which this helix rocking is tightly coupled to the substrate binding, to ensure strict 1:1 exchange. These results reveal the unique mechanism of sugar phosphate/phosphate exchange by TPT.

biochemistry↗

bioRxiv2017

VPS18 recruits VPS41 to the human HOPS complex via a RING-RING interaction

Eukaryotic cells use conserved multisubunit membrane tethering complexes, including CORVET and HOPS, to control the fusion of endomembranes. These complexes have been extensively studied in yeast, but to date there have been far fewer studies of metazoan CORVET and HOPS. Both of these complexes comprise six subunits: a common four-subunit core and two unique subunits. Once assembled, these complexes function to recognise specific endosomal membrane markers and facilitate SNARE-mediated membrane fusion. CORVET promotes the homotypic fusion of early endosomes, while HOPS promotes the fusion of lysosomes to late endosomes and autophagosomes. Many of the subunits of both CORVET and HOPS contain putative C-terminal zinc-finger domains. Here, the contribution of these domains to the assembly of the human CORVET and HOPS complexes has been examined. Using biochemical techniques, we demonstrate that the zinc-containing RING domains of human VPS18 and VPS41 interact directly to form a stable heterodimer. In cells, these RING domains are able to integrate into endogenous HOPS, showing that the VPS18 RING domain is required to recruit VPS41 to the core complex subunits. Importantly, this mechanism is not conserved throughout eukaryotes, as yeast Vps41 does not contain a C-terminal zinc-finger motif. The subunit analogous to VPS41 in human CORVET is VPS8, in which the RING domain has an additional C-terminal segment that is predicted to be disordered. Both the RING and disordered C-terminal domains are required for integration of VPS8 into endogenous CORVET complexes, suggesting that HOPS and CORVET recruit VPS41 and VPS8 via distinct molecular interactions.

biochemistry↗

bioRxiv2017

Chemical Gating of the Mechanosensitive Piezo1 Channel by Asymmetric Binding of its Agonist Yoda1

Piezo proteins are homotrimeric ion channels that play major roles in normal and pathological mechanotransduction signaling in mammalian organisms. Their pharmacological control hence represents a potential therapeutic avenue. Yoda1, a Piezo1-selective small molecule agonist, is the only known selective Piezo modulator. How Yoda1 selectively interacts with Piezo1 and opens its pore is unknown. Here, by engineering and characterizing chimeras, we identified a minimal region responsible for Yoda1 binding. This region is located at the interface between the pore and the putative mechanosensory domains in each subunit. By characterizing hybrid channels containing Yoda1-insensitive and Yoda1-sensitive monomers, we demonstrate that the presence of only one Yoda1-sensitive Piezo1 subunit is sufficient for chemical activation, implicating that the asymmetric binding of Yoda1 to a single subunit enables channel opening. These findings shed light onto the gating mechanisms of Piezo channels and will pave the way for the rationale design of new Piezo channels modulators.

biochemistry↗