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bioRxiv2016

Quantitative cross-linking/mass spectrometry using isotope-labeled cross-linkers and MaxQuant

AbstractThe conceptually simple step from cross-linking/mass spectrometry (CLMS) to quantitative cross-linking/mass spectrometry (QCLMS) is compounded by technical challenges. Currently, quantitative proteomics software is tightly integrated with the protein identification workflow. This prevents automatically quantifying other m/z features in a targeted manner including those associated with cross-linked peptides. Here we present a new release of MaxQuant that permits starting the quantification process from an m/z feature list. Comparing the automated quantification to a carefully manually curated test set of cross-linked peptides obtained by cross-linking C3 and C3b with BS3 and isotope-labeled BS3-d4 revealed a number of observations: 1) Fully automated process using MaxQuant can quantify cross-links in our reference dataset with 68% recall rate and 88% accuracy. 2) Hidden quantification errors can be converted into exposed failures by label-swap replica, which makes label-swap replica an essential part of QCLMS. 3) Cross-links that failed during automated quantification can be recovered by semi-automated re-quantification. The integrated workflow of MaxQuant and semi-automated assessment provides the maximum of quantified cross-links. In contrast, work on larger data sets or by less experienced users will benefit from full automation in MaxQuant.\n\nAbbreviations

Biochemistry↗

bioRxiv2016

Structure of complement C3(H2O) revealed by quantitative cross-linking/mass spectrometry and modeling

The slow but spontaneous and ubiquitous formation of C3(H2O), the hydrolytic and conformationally rearranged product of C3, initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. The structure of C3(H2O) has not been determined. Here we subjected C3(H2O) to quantitative cross-linking/mass spectrometry (QCLMS). This revealed details of the structural differences and similarities between C3(H2O) and C3, as well as between C3(H2O) and its pivotal proteolytic cleavage product, C3b, which shares functionally similarity with C3(H2O). Considered in combination with the crystal structures of C3 and C3b, the QCMLS data suggest that C3(H2O) generation is accompanied by the migration of the thioester-containing domain of C3 from one end of the molecule to the other. This creates a stable C3b-like platform able to bind the zymogen, factor B, or the regulator, factor H. Integration of available crystallographic and QCLMS data allowed the determination of a 3D model of the C3(H2O) domain architecture. The unique arrangement of domains thus observed in C3(H2O), which retains the anaphylatoxin domain (that is excised when C3 is enzymatically activated to C3b), can be used to rationalize observed differences between C3(H2O) and C3b in terms of complement activation and regulation.

Biochemistry↗

bioRxiv2016

High-throughput biochemical profiling reveals Cas9 off-target binding and unbinding heterogeneity

**. Introduction **. Introduction **. Main body **. Online Methods **. Author contributions **. Competing financial... ** References The bacterial adaptive immune system CRISPR-Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci1. To analyze Cas9s ability to bind DNA rapidly and specifically, we measured the kinetics of catalytically dead Cas9 (dCas9) interactions with a library of potential binding partners. Using a massively parallel assay of protein-DNA interactions derived from a high-throughput sequencing flow cell (HiTS-FLIP)2 and building on the established importance of protospacer adjacent motif (PAM) and seed recognition3- ...

Biochemistry↗

bioRxiv2016

Ubiquitin-mediated proteolysis in Xenopus extract

The small protein modifier, ubiquitin, can be covalently attached to proteins in the process of ubiquitylation, resulting in a variety of functional outcomes. In particular, the most commonly-associated and well-studied fate for proteins modified with ubiquitin is their ultimate destruction: degradation by the 26S proteasome via the ubiquitin-proteasome system, or digestion in lysosomes by proteolytic enzymes. From the earliest days of ubiquitylation research, a reliable and versatile \"cell-in-a-test-tube\" system has been employed in the form of cytoplasmic extracts from the eggs and embryos of the frog Xenopus laevis. Biochemical studies of ubiquitin and protein degradation using this system have led to significant advances particularly in the study of ubiquitin-mediated proteolysis, while the versatility of Xenopus as a developmental model has allowed investigation of the in vivo consequences of ubiquitylation. Here we describe the use and history of Xenopus extract in the study of ubiquitin-mediated protein degradation, and highlight the versatility of this system that has been exploited to uncover mechanisms and consequences of ubiquitylation and proteolysis.

Biochemistry↗

bioRxiv2016

Zika virus may not be alone: proteomics associates a bovine-like viral diarrhea virus to microcephaly

One Sentence SummaryProteomics analysis lead us to suspect the presence of a Bovine-like viral diarrhea virus (BVDV-like) in the brain tissue of fetuses bearing microcephaly during the outbreak in Paraiba State, Brazil, 2015.\n\nAbstractNo direct experimental causal evidence confirms that the Zika virus is the sole etiological agent responsible for the development of brain malformations in human fetuses during pregnancy. We used a discovery-driven approach to analyze protein extracts of three Zika positive brains. Shotgun mass spectrometry (MS) proteomics did not identify any Zika protein in all samples. However, MS detected the presence of peptide(s) from the polyprotein of a Bovine-like viral diarrhea virus (BVDV-like) in Zika-positive brains. These results indicate that Zika virus may not be, per se, the only etiological agent responsible for microcephaly and suggests that discovery-driven approaches play an essential role in the screening of fluids or tissues for virus or other etiological agents.

Biochemistry↗

bioRxiv2016

Antiparallel protocadherin homodimers use distinct affinity- and specificity mediating regions in cadherin repeats 1-4

Protocadherins (Pcdhs) are cell adhesion and signaling proteins used by neurons to develop and maintain neuronal networks, relying on trans homophilic interactions between their extracellular cadherin (EC) repeat domains. We present the structure of the antiparallel EC1-4 homodimer of human PcdhYB3, a member of the {gamma} subfamily of clustered Pcdhs. Structure and sequence comparisons of , {beta}, and {gamma} clustered Pcdh isoforms illustrate that subfamilies encode specificity in distinct ways through diversification of loop region structure and composition in EC2 and EC3, which contains isoform-specific conservation of primarily polar residues. In contrast, the EC1/EC4 interface comprises hydrophobic interactions that provide non-selective dimerization affinity. Using sequence coevolution analysis, we found evidence for a similar antiparallel EC1-4 interaction in non-clustered Pcdh families. We thus deduce that the EC1-4 antiparallel homodimer is a general interaction strategy that evolved before the divergence of these distinct protocadherin families.

Biochemistry↗

bioRxiv2016

Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments

Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage. We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid. We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase centre at a dimer interface. EncFtn adopts an open decameric structure that is topologically distinct from other ferritins. While EncFtn acts as a ferroxidase, it cannot mineralize iron. Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage. This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system where the oxidation and mineralization of iron are distributed between two proteins.

Biochemistry↗

bioRxiv2016

The metabolic profile of lag and exponential phases of Saccharomyces cerevisiae growth changes continuously

Cellular growth is usually separated in well-defined phases. For microorganism like Saccharomyces cerevisiae, two phases usually defined are (1) a lag phase, in which no growth is observed and cells adapt to a new environment, followed by (2) an exponential phase, in which rapid proliferation occurs. Here we investigate whether these well-defined phases are uniform. By using flow-microcalorimetry, we found that the metabolic profile of the culture is continuously changing, both in the lag and exponential phases of growth. Along the lag phase there is a continuous increase in the energy that is dissipated irreversibly as heat, while in the exponential phase the opposite occurs. We also confirm recent observations that the oxidative component of metabolism decreases along the exponential phase. Interestingly, nutrient limitation further decreases the amount of energy that is dissipated irreversibly. Altogether, this points to a picture in which cells respond rapidly to minute environmental changes by adjusting their metabolic profile.

Biochemistry↗

bioRxiv2016

Architectures of a lipid transport systems for the bacterial outer membrane

How phospholipids are trafficked between the bacterial inner and outer membranes through the intervening hydrophilic space of the periplasm is not known. Here we report that members of the mammalian cell entry (MCE) protein family form structurally diverse hexameric rings and barrels with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring as part of a larger ABC transporter complex in the inner membrane, and employs a soluble lipid-binding protein to ferry lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the entire periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the inner and outer membranes of bacteria and some eukaryotic organelles.\n\nHIGHLIGHTSO_LIMCE proteins adopt diverse architectures for transporting lipids across the bacterial periplasm\nC_LIO_LICryo-EM and X-ray structures reveal how the MlaFEDB complex, along with MlaC, might shuttle lipids across the periplasm\nC_LIO_LI3.9 [A] cryo-EM structure of PqiB reveals a syringe-like architecture with a continuous central channel\nC_LIO_LIYebT forms a a segmented tube-like structure, and YebT and PqiB are poised to directly link the inner and outer membranes to facilitate lipid transport.\nC_LI

Biochemistry↗

bioRxiv2016

The Origin of Coupled Chloride and Proton Transport in a Cl-/H+ Antiporter

The ClC family of transmembrane proteins functions throughout nature to control the transport of Cl- ions across biological membranes. ClC-ec1 from Escherichia coli is an antiporter, coupling the transport of Cl- and H+ ions in opposite directions and driven by the concentration gradients of the ions. Despite keen interest in this protein, the molecular mechanism of the Cl-/H+ coupling has not been fully elucidated. Here, we have used multiscale simulation to help identify the essential mechanism of the Cl-/H+ coupling. We find that the highest barrier for proton transport (PT) from the intra- to extracellular solution is attributable to a chemical reaction--the deprotonation of glutamic acid 148 (E148). This barrier is significantly reduced by the binding of Cl- in the \"central\" site (Cl-cen), which displaces E148 and thereby facilitates its deprotonation. Conversely, in the absence of Cl-cen E148 favors the \"down\" conformation, which results in a much higher cumulative rotation and deprotonation barrier that effectively blocks PT to the extracellular solution. Thus, the rotation of E148 plays a critical role in defining the Cl-/H+ coupling. As a control, we have also simulated PT in the ClC-ec1 E148A mutant to further understand the role of this residue. Replacement with a non-protonatable residue greatly increases the free energy barrier for PT from E203 to the extracellular solution, explaining the experimental result that PT in E148A is blocked whether or not Cl-cen is present. The results presented here suggest both how a chemical reaction can control the rate of PT and also how it can provide a mechanism for a coupling of the two ion transport processes.

Biochemistry↗

bioRxiv2016

A Rationally Designed Aminoacyl-tRNA Synthetase for Genetically Encoded Fluorescent Amino Acids

The incorporation of non-canonical amino acids into proteins has emerged as a promising strategy to manipulate and study protein structure-function relationships with superior precision in vitro and in vivo. To date, fluorescent non-canonical amino acids (f-ncAA) have been successfully incorporated in proteins expressed in bacterial systems, Xenopus oocytes, and HEK-293T cells. Here, we describe the rational generation of an orthogonal aminoacyltRNA synthetase based on the E. coli tyrosine synthetase that is capable of encoding the f-ncAA tyr-coumarin in HEK-293T cells.

Biochemistry↗

bioRxiv2016

Design: An assay based on single-polypeptide-chain heterodimeric A2AR/D2R and non-oligomerized fusions for in vivo analysis of their allosteric receptor-receptor interactions

BackgroundThe adenosine A2A receptor (A2AR) heteromerizes with the dopamine D2 receptor (D2R). In order to explore their functional interaction, we engineered previously stable single-polypeptide-chain (sc) A2AR/D2LR: whether the molecular entity of the striatal A2AR/D2R antagonism, i.e., scA2AR/D2Rs are just A2AR/D2R with the antagonism, remains unresolved.\n\nNew MethodTo further clarify the heteromerization through the scA2AR/D2LR, we here designed supramolecularly exclusive monomers and dimers, using the C{varepsilon}2 domain of IgE-Fc or apoproteins of the bacterial light-harvesting antenna complex.\n\nResultsA concept of the recptor protein assembly regulation, i.e., the selective monomer/non-obligate dimer formation was obtained. Although none of these new fusions were constructed or tested, we could aim at obtaining heterodimer-specific agents, using the scA2AR/D2R. Whether the resulting designs were explained feasibly and rationally was addressed. The structure and function of the non-obligate dimer were here discussed through scA2AR/D2R, focusing on the procedure of the membrane protein design and methods for transient protein-protein interactions.\n\nSummary and OutlookGiven that upon being expressed and allosteric regulation occurs regardless of specific signal to non-specific noise (S/N) ratio, the supramolecular designs, allowing us to express selectively monomer/non-obligate dimer of class A GPCR, are experimentally testable and will be used to confirm in vivo that such low S/N ratio interaction between A2AR and D2LR functions in the dopamine neurotransmission in the striatum.\n\nAbbreviations

Biochemistry↗

bioRxiv2016

Beyond the myosin mesa: a potential unifying hypothesis on the underlying molecular basis of hyper-contractility caused by a majority of hypertrophic cardiomyopathy mutations

AbstractHypertrophic cardiomyopathy (HCM), the most commonly occurring inherited cardiovascular disease, is primarily caused by mutations in human {beta}-cardiac myosin and myosin binding protein-C. It has been thought that such mutations in myosin increase the intrinsic force of the motor, its velocity of contraction, or its ATPase activity, giving rise to hyper-contractility. We hypothesize that while these parameters are mildly affected by most myosin HCM-causing mutations, a major effect of a majority of myosin HCM mutations is likely to involve an increase in the number of myosin heads that are functionally accessible (Na) for interaction with actin in the sarcomere. We consider a model involving three types of interactions involving the myosin mesa and the converter domain of the myosin motor that hold myosin heads in a sequestered state, likely to be released in a graded manner as the demands on the heart increase: 1) the two myosin heads binding to one another, 2) one head binding to its own coiled-coil tail, and 3) the other head binding to myosin binding protein-C. In addition there is clear evidence of interaction between the coiled-coil tail of myosin and myosin binding protein-C. Experimentally, here we focus on myosin head binding to its own coiled-coil tail and to myosin binding protein-C. We show that phosphorylation of the myosin regulatory light chain and myosin binding protein-C weaken these respective associations, consistent with known enhancements of sarcomere function by these phosphorylations. We show that these interactions are weakened as a result of myosin HCM mutations, in a manner consistent with our structural model. Our data suggests a potential unifying hypothesis for the molecular basis of hyper-contractility caused by human hypertrophic cardiomyopathy myosin mutations, whereby the mutations give rise to an increase in the number of myosin heads that are functionally accessible for interaction with actin in the sarcomere, causing the hyper-contractility observed clinically.

Biochemistry↗

bioRxiv2016

In vivo mapping of tissue- and subcellular-specific proteomes in Caenorhabditis elegans

Multicellular organisms are composed of tissues that have distinct functions requiring specialized proteomes. To define the proteome of a live animal with tissue and subcellular resolution, we adapted a localized proteomics technology for use in the multicellular model organism Caenorhabditis elegans. This approach couples tissue- and location-specific expression of the enzyme ascorbate peroxidase (APX), which facilitates proximity-based protein labeling in vivo, and quantitative proteomics to identify tissue- and subcellular-restricted proteomes. We identified and localized over 3000 proteins from strains of C. elegans expressing APX in either the nucleus or cytoplasm of the intestine, epidermis, body wall muscle, or pharyngeal muscle. We also identified several hundred proteins that were specifically localized to one of the four tissues analyzed or specifically localized to the cytoplasm or the nucleus. This approach resulted in the identification of both previously characterized and unknown nuclear and cytoplasmic proteins. Further, we confirmed the tissue- and subcellular-specific localization of a subset of identified proteins using GFP-tagging and fluorescence microscopy, validating our in vivo proximity-based proteomics technique. Together, these results demonstrate a new approach that enables the tissue- and subcellular-specific identification and quantification of proteins within a live animal.

Biochemistry↗

bioRxiv2016

On the estimation errors of KM and V from time-course experiments using the Michaelis-Menten equation

The conditions under which the Michaelis-Menten equation accurately captures the steady-state kinetics of a simple enzyme-catalyzed reaction is contrasted with the conditions under which the same equation can be used to estimate parameters, KM and V, from progress curve data. Validity of the underlying assumptions leading to the Michaelis-Menten equation are shown to be necessary, but not sufficient to guarantee accurate estimation of KM and V. Detailed error analysis and numerical \"experiments\" show the required experimental conditions for the independent estimation of both KM and V from progress curves. A timescale, tQ, measuring the portion of the time course over which the progress curve exhibits substantial curvature provides a novel criterion for accurate estimation of KM and V from a progress curve experiment. It is found that, if the initial substrate concentration is of the same order of magnitude as KM, the estimated values of the KM and V will correspond to their true values calculated from the microscopic rate constants of the corresponding mass-action system, only so long as the initial enzyme concentration is less than KM.

Biochemistry↗

bioRxiv2016

How the AFF1/4 scaffold recruits the elongation factor ELL2 to promote HIV-1 proviral transcription

The intrinsically disordered scaffold proteins AFF1/4 and the transcription elongation factors ELL1/2 are core components of the superelongation complex required for HIV-1 proviral transcription. We determined the 2.0-[A] resolution crystal structure of the human ELL2 C-terminal domain bound to its 50-residue binding site on AFF4, the ELLBow. The ELL2 domain has the same arch-shaped fold as the tight junction protein occludin. The ELLBow consists of an N-terminal helix followed by an extended hairpin that we refer to as the elbow joint, and occupies most of the concave surface of ELL2. This surface is important for the ability of ELL2 to promote HIV-1 Tat-mediated proviral transcription. The AFF4-ELL2 interface is imperfectly packed, leaving a cavity suggestive of a potential binding site for transcription-promoting small molecules.

Biochemistry↗

bioRxiv2016

Multiple binding modes of ibuprofen in human serum albumin identified by absolute binding free energy calculations

Human serum albumin possesses multiple binding sites and transports a wide range of ligands that include the anti-inflammatory drug ibuprofen. A complete map of the binding sites of ibuprofen in albumin is difficult to obtain in traditional experiments, because of the structural adaptability of this protein in accommodating small ligands. In this work, we provide a set of predictions covering the geometry, affinity of binding and protonation state for the pharmaceutically most active form (S- isomer) of ibuprofen to albumin, by using absolute binding free energy calculations in combination with classical molecular dynamics (MD) simulations and molecular docking. The most favorable binding modes correctly reproduce several experimentally identified binding locations, which include the two Sudlows drug sites (DS2 and DS1) and the fatty acid binding sites 6 and 2 (FA6 and FA2). Previously unknown details of the binding conformations were revealed for some of them, and formerly undetected binding modes were found in other protein sites. The calculated binding affinities exhibit trends which seem to agree with the available experimental data, and drastically degrade when the ligand is modeled in a protonated (neutral) state, indicating that ibuprofen associates with albumin preferentially in its charged form. These findings provide a detailed description of the binding of ibuprofen, help to explain a wide range of results reported in the literature in the last decades, and demonstrate the possibility of using simulation methods to predict ligand binding to albumin.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC=\"FIGDIR/small/068502_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (31K):\norg.highwire.dtl.DTLVardef@1b04be9org.highwire.dtl.DTLVardef@147e748org.highwire.dtl.DTLVardef@1a13803org.highwire.dtl.DTLVardef@164624f_HPS_FORMAT_FIGEXP M_FIG C_FIG FocusAlchemical free energy methods can identify favored binding modes of a ligand within a large protein with multiple binding sites\n\nHighlightsO_LIHuman serum albumin binds the anti-inflammatory drug ibuprofen in multiple sites\nC_LIO_LIAlchemical free energy calculations predicted favored binding modes of ibuprofen\nC_LIO_LIBound geometry, affinity and protonation state of the ligand were determined\nC_LIO_LISimulations identified a number of previously undetected binding sites for ibuprofen\nC_LIO_LIFree energy methods can be used to study large proteins with multiple binding sites\nC_LI

Biochemistry↗

bioRxiv2016

Development and Application of Equilibrium Thermodynamic Models Describing Coupled Protein Oligomerization and Ligand Binding

The association and dissociation of protein oligomers is frequently coupled to the binding of ligands, facilitating the regulation of many biological processes. Equilibrium thermodynamic models are needed to describe the linkage between ligand binding and homo-oligomerization. These models must be parameterized in a way that makes physical interpretation straightforward, and allows elaborations or simplifications to be readily incorporated. We propose a systematic framework for the equilibrium analysis of ligand-linked oligomerization, treating in detail the case of a homo-oligomer with cyclic point group symmetry, where each subunit binds a ligand at a single site. Exploiting the symmetry of the oligomer, in combination with a nearest-neighbors approximation, we derive a class of site-specific ligand binding models involving only four parameters, irrespective of the size of the oligomer. The model parameters allow direct quantitative assessment of ligand binding cooperativity, and the influence of ligand binding on protein oligomerization, which are the key questions of biological interest. These models, which incorporate multiple types of linkage, are practically applicable, and we show how Markov Chain Monte Carlo (MCMC) methods can be used to characterize the agreement of the model with experimental data. Simplifications to the model emerge naturally, as its parameters take on extremal values. The nearest-neighbors approximation underpinning the model is transparent, and the model could be augmented in obvious fashion if the approximation were inadequate. The approach is generalizable, and could be used to treat more complex situations, involving more than a single kind of ligand, or a different protein symmetry.\n\nAuthor SummaryThe assembly and disassembly of protein complexes in response to the binding of ligands is a ubiquitous biological phenomenon. This is often linked, in turn, to the activation or deactivation of protein function. Methods are therefore needed to quantitate the linkage or coupling between protein assembly and effector binding, requiring the development of mathematical models describing the coupled binding processes. As proteins usually assemble in a symmetric fashion, the nature of any symmetry present has to be considered during model construction. We have developed a class of models than can effectively describe the coupling between effector binding and protein assembly into symmetric ring-like structures of any size. Despite the relatively complex mathematical form of the models, they are practically applicable. Markov Chain Monte Carlo methods, a form of Bayesian statistical analysis, can be used to analyze the fit of the models to experimental data, and recover the model parameters. This allows the direct assessment of the nature and magnitude of the coupling between effector binding and protein assembly, as well as other relevant characteristics of the system.

Biochemistry↗