ROR and RYK extracellular region structures suggest that receptor tyrosine kinases have distinct WNT-recognition modes.

WNTs play key roles in development and disease, signaling through Frizzled (FZD) seven-pass transmembrane receptors and numerous co-receptors including ROR and RYK family receptor tyrosine kinases (RTKs). We describe crystal structures and WNT-binding characteristics of extracellular regions from the Drosophila ROR and RYK orthologs Nrk (neurospecific receptor tyrosine kinase) and Derailed-2 (Drl-2), which bind WNTs though a FZD-related cysteine-rich domain (CRD) and WNT-inhibitory factor (WIF) domain respectively. Our crystal structures suggest that neither Nrk nor Drl-2 can accommodate the acyl chain typically attached to WNTs. The Nrk CRD contains a deeply buried bound fatty acid, unlikely to be exchangeable. The Drl-2 WIF domain lacks the lipid-binding site seen in WIF-1. We also find that recombinant DWnt-5 can bind Drosophila ROR and RYK orthologs despite lacking an acyl chain. Alongside analyses of WNT/receptor interaction sites, our structures provide further insight into how WNTs may recruit RTK co-receptors into signaling complexes.

Development and validation of ELISA method for detection of human signal regulatory protein α-anti-CD20 mouse chimeric antibody fusion protein IMM0306 in human serum / 药学学报

IMM0306 is a recombinant human signal regulatory protein α-anti-CD20 mouse chimeric antibody fusion protein, intended to treat refractory or recurrent CD20 positive B-cell non-Hodgkin lymphoma (B-NHL) in clinical. In this study, an ELISA method was established to evaluate the pharmacokinetics of IMM0306 in human serum. The experiment was approved by the Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences (No. CTR20192612). Recombinant human CD47 protein was coated with the plate overnight, blocking the plate with 5% skin milk for 2 h. After washing, 100 μL per well standard and unknown samples were added, and incubated for 1.5 h. After washing, the detection antibody Anti-IMM0306-Biotin was added and incubated for 1 h, and then HRP-labeled streptavidin was added for 1 h, and the color was detected. The optimal concentration of coating reagent was 2 μg·mL-1 by ELISA method, and the optimal dilution of anti-IMM0306-biotin and SA-HRP were 1∶500 and 1∶5 000, respectively. The lower limit of quantitation was 4 ng·mL-1, and the standard curve range was 4-100 ng·mL-1. The verification results of the method meets the corresponding acceptance criteria, and can be used in IMM0306 clinical pharmacokinetic studies.

Structural and mechanistic insights into Hsp104 function revealed by synchrotron X-ray footprinting.

Hsp104 is a hexameric AAA+ ring translocase, which drives protein disaggregation in nonmetazoan eukaryotes. Cryo-EM structures of Hsp104 have suggested potential mechanisms of substrate translocation, but precisely how Hsp104 hexamers disaggregate proteins remains incompletely understood. Here, we employed synchrotron X-ray footprinting to probe the solution-state structures of Hsp104 monomers in the absence of nucleotide and Hsp104 hexamers in the presence of ADP or ATPγS (adenosine 5'-O-(thiotriphosphate)). Comparing side-chain solvent accessibilities between these three states illuminated aspects of Hsp104 structure and guided design of Hsp104 variants to probe the disaggregase mechanism in vitro and in vivo We established that Hsp104 hexamers switch from a more-solvated state in ADP to a less-solvated state in ATPγS, consistent with switching from an open spiral to a closed ring visualized by cryo-EM. We pinpointed critical N-terminal domain (NTD), NTD-nucleotide-binding domain 1 (NBD1) linker, NBD1, and middle domain (MD) residues that enable intrinsic disaggregase activity and Hsp70 collaboration. We uncovered NTD residues in the loop between helices A1 and A2 that can be substituted to enhance disaggregase activity. We elucidated a novel potentiated Hsp104 MD variant, Hsp104-RYD, which suppresses α-synuclein, fused in sarcoma (FUS), and TDP-43 toxicity. We disambiguated a secondary pore-loop in NBD1, which collaborates with the NTD and NBD1 tyrosine-bearing pore-loop to drive protein disaggregation. Finally, we defined Leu-601 in NBD2 as crucial for Hsp104 hexamerization. Collectively, our findings unveil new facets of Hsp104 structure and mechanism. They also connect regions undergoing large changes in solvation to functionality, which could have profound implications for protein engineering.

ExMS2: An Integrated Solution for Hydrogen-Deuterium Exchange Mass Spectrometry Data Analysis.

Hydrogen-deuterium exchange mass spectrometry (HDX MS) has become an important technique for the analysis of protein structure and dynamics. Data analysis remains a bottleneck in the workflow. Sophisticated computer analysis is required to scan through the voluminous MS output in order to find, identify, and validate many partially deuterated peptides, elicit the HDX information, and extend the results to higher structural resolution. We previously made available two software suites, ExMS for identification and analysis of peptide isotopic envelopes in the HDX MS raw data and HDsite for residue-level resolution. Further experience has led to advances in the usability and performance of both programs. Also, newly added modules deal with ETD/ECD analysis, multimodal mass spectra analysis, and presentation options. These advances have been integrated into a stand-alone software solution named ExMS2. The package has been successfully tested by many workers in fine scale epitope mapping, in protein folding studies, and in dissecting structure and structure change of large protein complexes. A description and tutorial for this major upgrade are given here.

Hydrogen-Deuterium Exchange Coupled to Top- and Middle-Down Mass Spectrometry Reveals Histone Tail Dynamics before and after Nucleosome Assembly.

Until recently, a major limitation of hydrogen-deuterium exchange mass spectrometry (HDX-MS) was that resolution of deuterium localization was limited to the length of the peptide generated during proteolysis. However, electron transfer dissociation (ETD) has been shown to preserve deuterium label in the gas phase, enabling better resolution. To date, this technology remains mostly limited to small, already well-characterized proteins. Here, we optimize, expand, and adapt HDX-MS tandem MS (MS/MS) capabilities to accommodate histone and nucleosomal complexes on top-down HDX-MS/MS and middle-down HDX-MS/MS platforms and demonstrate that near site-specific resolution of deuterium localization can be obtained with high reproducibility. We are able to study histone tail dynamics in unprecedented detail, which have evaded analysis by traditional structural biology techniques for decades, revealing important insights into chromatin biology. Together, the results of these studies highlight the versatility, reliability, and reproducibility of ETD-based HDX-MS/MS methodology to interrogate large protein and protein/DNA complexes.

Folding of maltose binding protein outside of and in GroEL.

We used hydrogen exchange-mass spectrometry (HX MS) and fluorescence to compare the folding of maltose binding protein (MBP) in free solution and in the GroEL/ES cavity. Upon refolding, MBP initially collapses into a dynamic molten globule-like ensemble, then forms an obligatory on-pathway native-like folding intermediate (1.2 seconds) that brings together sequentially remote segments and then folds globally after a long delay (30 seconds). A single valine to glycine mutation imposes a definable folding defect, slows early intermediate formation by 20-fold, and therefore subsequent global folding by approximately twofold. Simple encapsulation within GroEL repairs the folding defect and reestablishes fast folding, with or without ATP-driven cycling. Further examination exposes the structural mechanism. The early folding intermediate is stabilized by an organized cluster of 24 hydrophobic side chains. The cluster preexists in the collapsed ensemble before the H-bond formation seen by HX MS. The V9G mutation slows folding by disrupting the preintermediate cluster. GroEL restores wild-type folding rates by restabilizing the preintermediate, perhaps by a nonspecific equilibrium compression effect within its tightly confining central cavity. These results reveal an active GroEL function other than previously proposed mechanisms, suggesting that GroEL possesses different functionalities that are able to relieve different folding problems. The discovery of the preintermediate, its mutational destabilization, and its restoration by GroEL encapsulation was made possible by the measurement of a previously unexpected type of low-level HX protection, apparently not dependent on H-bonding, that may be characteristic of proteins in confined spaces.

Protein Folding-How and Why: By Hydrogen Exchange, Fragment Separation, and Mass Spectrometry.

Advanced hydrogen exchange (HX) methodology can now determine the structure of protein folding intermediates and their progression in folding pathways. Key developments over time include the HX pulse labeling method with nuclear magnetic resonance analysis, the fragment separation method, the addition to it of mass spectrometric (MS) analysis, and recent improvements in the HX MS technique and data analysis. Also, the discovery of protein foldons and their role supplies an essential interpretive link. Recent work using HX pulse labeling with MS analysis finds that a number of proteins fold by stepping through a reproducible sequence of native-like intermediates in an ordered pathway. The stepwise nature of the pathway is dictated by the cooperative foldon unit construction of the protein. The pathway order is determined by a sequential stabilization principle; prior native-like structure guides the formation of adjacent native-like structure. This view does not match the funneled energy landscape paradigm of a very large number of folding tracks, which was framed before foldons were known and is more appropriate for the unguided residue-level search to surmount an initial kinetic barrier rather than for the overall unfolded-state to native-state folding pathway.

Cytochrome c folds through foldon-dependent native-like intermediates in an ordered pathway.

Previous hydrogen exchange (HX) studies of the spontaneous reversible unfolding of Cytochrome c (Cyt c) under native conditions have led to the following conclusions. Native Cyt c (104 residues) is composed of five cooperative folding units, called foldons. The high-energy landscape is dominated by an energy ladder of partially folded forms that differ from each other by one cooperative foldon unit. The reversible equilibrium unfolding of native Cyt c steps up through these intermediate forms to the unfolded state in an energy-ordered sequence, one foldon unit at a time. To more directly study Cyt c intermediates and pathways during normal energetically downhill kinetic folding, the present work used HX pulse labeling analyzed by a fragment separation-mass spectrometry method. The results show that 95% or more of the Cyt c population folds by stepping down through the same set of foldon-dependent pathway intermediates as in energetically uphill equilibrium unfolding. These results add to growing evidence that proteins fold through a classical pathway sequence of native-like intermediates rather than through a vast number of undefinable intermediates and pathways. The present results also emphasize the condition-dependent nature of kinetic barriers, which, with less informative experimental methods (fluorescence, etc.), are often confused with variability in intermediates and pathways.

High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome.

Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen-deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention.

Telomere G-Quadruplex as a Potential Target to Accelerate Telomere Shortening by Expanding the Incomplete End-Replication of Telomere DNA.

Chromosomes in human cells are protected by telomeres. Telomere shortens during each round of cell division because of the DNA end-replication problem. Cancer cells maintain telomere length homeostasis by either telomerase or/and the alternative lengthening of telomere (ALT) mechanism to sustain their division potential. Telomeric DNA tends to form G-quadruplex preferentially at the extreme 3' end. This unique feature prevents the 3' end from being used as a substrate of telomerase and as a primer in the ALT. Therefore, stabilizing telomere G-quadruplex is expected to inhibit both pathways and limit the proliferation of cancer cells. Based on a mathematical modeling and experimental results, this mini-review proposes a hypothesis that the formation of G-quadruplex in telomere may constitute a significant contribution to the incomplete end-replication of telomere DNA by preventing the priming of DNA synthesis near the 3' end during telomere replication. According to this, stabilization of telomere G-quadruplex by chemical ligand may promise to accelerate telomere shortening in proliferating cells.

Determination of γ-aminobutyric acid in human plasma by LC-MS/MS and its preliminary application to a human pharmacokinetic study / 药学学报

A rapid, sensitive and convenient LC-MS/MS method was developed for the determination of γ-aminobutyric acid (GABA) in human plasma. d2-γ-Aminobutyric acid (d2-GABA) was synthesized as internal standard (IS). After extraction from human plasma by protein precipitation with acetonitrile, all analytes were separated on a Luna HILIC column (100 mm x 3.0 mm, 3 μm) using an isocratic mobile phase of water: acetonitrile: formic acid (20 : 80 : 0.12) with a flow rate of 0.5 mL x min(-1). Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode (MRM) in positive electrospray ionization using the transitions of m/z 104 --> 69 for GABA and m/z 106 --> 71 for d2-GABA. The method was linear in the concentration range of 5.00 to 1 000 ng x mL(-1). The intra- and inter-day precisions were within 9.9%, and accuracy ranged from 99.1% to 104%, within the acceptable limit across all concentrations. The method was successfully applied to a pharmacokinetic study of GABA tablets in healthy Chinese volunteers.

Determination of γ-aminobutyric acid in human plasma by LC-MS/MS and its preliminary application to a human pharmacokinetic study / 药学学报

A rapid, sensitive and convenient LC-MS/MS method was developed for the determination of γ-aminobutyric acid (GABA) in human plasma. d2-γ-Aminobutyric acid (d2-GABA) was synthesized as internal standard (IS). After extraction from human plasma by protein precipitation with acetonitrile, all analytes were separated on a Luna HILIC column (100 mm x 3.0 mm, 3 μm) using an isocratic mobile phase of water: acetonitrile: formic acid (20 : 80 : 0.12) with a flow rate of 0.5 mL x min(-1). Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode (MRM) in positive electrospray ionization using the transitions of m/z 104 --> 69 for GABA and m/z 106 --> 71 for d2-GABA. The method was linear in the concentration range of 5.00 to 1 000 ng x mL(-1). The intra- and inter-day precisions were within 9.9%, and accuracy ranged from 99.1% to 104%, within the acceptable limit across all concentrations. The method was successfully applied to a pharmacokinetic study of GABA tablets in healthy Chinese volunteers.

Protein hydrogen exchange at residue resolution by proteolytic fragmentation mass spectrometry analysis.

Hydrogen exchange technology provides a uniquely powerful instrument for measuring protein structural and biophysical properties, quantitatively and in a nonperturbing way, and determining how these properties are implemented to produce protein function. A developing hydrogen exchange-mass spectrometry method (HX MS) is able to analyze large biologically important protein systems while requiring only minuscule amounts of experimental material. The major remaining deficiency of the HX MS method is the inability to deconvolve HX results to individual amino acid residue resolution. To pursue this goal we used an iterative optimization program (HDsite) that integrates recent progress in multiple peptide acquisition together with previously unexamined isotopic envelope-shape information and a site-resolved back-exchange correction. To test this approach, residue-resolved HX rates computed from HX MS data were compared with extensive HX NMR measurements, and analogous comparisons were made in simulation trials. These tests found excellent agreement and revealed the important computational determinants.

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry.

The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis by an advanced fragment separation mass spectrometry technology. The results show that folding proceeds through distinct intermediates in a stepwise pathway that sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as a concerted transition of one or more segments from an HX-unprotected to an HX-protected state. Deconvolution of the data to near amino acid resolution shows that each step corresponds to the folding of a secondary structural element of the native protein, termed a "foldon." Each folded segment is retained through subsequent steps of foldon addition, revealing a stepwise buildup of the native structure via a single dominant pathway. Analysis of the pertinent literature suggests that this model is consistent with experimental results for many proteins and some current theoretical results. Two biophysical principles appear to dictate this behavior. The principle of cooperativity determines the central role of native-like foldon units. An interaction principle termed "sequential stabilization" based on native-like interfoldon interactions orders the pathway.

Apolipoprotein A-I helical structure and stability in discoidal high-density lipoprotein (HDL) particles by hydrogen exchange and mass spectrometry.

To understand high-density lipoprotein (HDL) structure at the molecular level, the location and stability of α-helical segments in human apolipoprotein (apo) A-I in large (9.6 nm) and small (7.8 nm) discoidal HDL particles were determined by hydrogen-deuterium exchange (HX) and mass spectrometry methods. The measured HX kinetics of some 100 apoA-I peptides specify, at close to amino acid resolution, the structural condition of segments throughout the protein sequence and changes in structure and stability that occur on incorporation into lipoprotein particles. When incorporated into the large HDL particle, the nonhelical regions in lipid-free apoA-I (residues 45-53, 66-69, 116-146, and 179-236) change conformation from random coil to α-helix so that nearly the entire apoA-I molecule adopts helical structure (except for the terminal residues 1-6 and 237-243). The amphipathic α-helices have relatively low stability, in the range 3-5 kcal/mol, indicating high flexibility and dynamic unfolding and refolding in seconds or less. A segment encompassed by residues 125-158 exhibits bimodal HX labeling indicating co-existing helical and disordered loop conformations that interchange on a time scale of minutes. When incorporated around the edge of the smaller HDL particle, the increase in packing density of the two apoA-I molecules forces about 20% more residues out of direct contact with the phospholipid molecules to form disordered loops, and these are the same segments that form loops in the lipid-free state. The region of disc-associated apoA-I that binds the lecithin-cholesterol acyltransferase enzyme is well structured and not a protruding unstructured loop as reported by others.

Many overlapping peptides for protein hydrogen exchange experiments by the fragment separation-mass spectrometry method.

Measurement of the naturally occurring hydrogen exchange (HX) behavior of proteins can in principle provide highly resolved thermodynamic and kinetic information on protein structure, dynamics, and interactions. The HX fragment separation-mass spectrometry method (HX-MS) is able to measure hydrogen exchange in biologically important protein systems that are not accessible to NMR methods. In order to achieve high structural resolution in HX-MS experiments, it will be necessary to obtain many sequentially overlapping peptide fragments and be able to identify and analyze them efficiently and accurately by mass spectrometry. This paper describes operations which, when applied to four different proteins ranging in size from 140 to 908 residues, routinely provides hundreds of useful unique peptides, covering the entire protein length many times over. Coverage in terms of the average number of peptide fragments that span each amino acid exceeds 10. The ability to achieve these results required the integrated application of experimental methods that are described here and a computer analysis program, called ExMS, described in a following paper.

ExMS: data analysis for HX-MS experiments.

A previous paper considered the problems that presently limit the hydrogen exchange-mass spectrometry (HX-MS) method for studying the biophysical and functional properties of proteins. Many of these problems can be overcome by obtaining and analyzing hundreds of sequentially overlapping peptide fragments that cover the protein many times over (Mayne et al. J. Am. Soc. Mass Spectrom. 2011: 10.1007/s13361-011-0235-4). This paper describes a computer program called ExMS that furthers this advance by making it possible to efficiently process crowded mass spectra and definitively identify and characterize these many peptide fragments. ExMS automatically scans through high resolution MS data to find the individual isotopic peaks and isotopic envelopes of a list of peptides previously identified by MS/MS. It performs a number of tests to ensure correct identification in spite of peptide overlap in both chromatographic and mass spectrometric dimensions and possible multi-modal envelopes due to static or dynamic structural heterogeneity or HX EX1 behavior. The program can automatically process data from many sequential HX time points with no operator intervention at the rate of ~2 sec per peptide per HX time point using desktop computer equipment, but it also provides for rapid manual checking and decision when ambiguity exists. Additional subroutines can provide a step by step report of performance at each test along the way and parameter adjustment, deconvolute isotopic envelopes, and plot the time course of single and multi-modal H-D exchange. The program will be available on an open source basis at: http://HX2.med.upenn.edu/download.html.

Replacement of histone H3 with CENP-A directs global nucleosome array condensation and loosening of nucleosome superhelical termini.

Centromere protein A (CENP-A) is a histone H3 variant that marks centromere location on the chromosome. To study the subunit structure and folding of human CENP-A-containing chromatin, we generated a set of nucleosomal arrays with canonical core histones and another set with CENP-A substituted for H3. At the level of quaternary structure and assembly, we find that CENP-A arrays are composed of octameric nucleosomes that assemble in a stepwise mechanism, recapitulating conventional array assembly with canonical histones. At intermediate structural resolution, we find that CENP-A-containing arrays are globally condensed relative to arrays with the canonical histones. At high structural resolution, using hydrogen-deuterium exchange coupled to mass spectrometry (H/DX-MS), we find that the DNA superhelical termini within each nucleosome are loosely connected to CENP-A, and we identify the key amino acid substitution that is largely responsible for this behavior. Also the C terminus of histone H2A undergoes rapid hydrogen exchange relative to canonical arrays and does so in a manner that is independent of nucleosomal array folding. These findings have implications for understanding CENP-A-containing nucleosome structure and higher-order chromatin folding at the centromere.

Kinetic and thermodynamic control of G-quadruplex folding.

A matter of speed: when allowed to fold in a K(+)/poly(ethylene glycol) solution, the guanine (G)-rich strand of vertebrate telomere DNA forms a parallel/antiparallel G-quadruplex, which is a (3+1) hybrid, within microseconds before slowly transforming into the parallel one within hours. Thus, the conformation that a G-quadruplex initially adopts under physiological conditions may not be the one it adopts at the equilibrium state.

G-quadruplex preferentially forms at the very 3' end of vertebrate telomeric DNA.

Human chromosome ends are protected with kilobases repeats of TTAGGG. Telomere DNA shortens at replication. This shortening in most tumor cells is compensated by telomerase that adds telomere repeats to the 3' end of the G-rich telomere strand. Four TTAGGG repeats can fold into G-quadruplex that is a poor substrate for telomerase. This property has been suggested to regulate telomerase activity in vivo and telomerase inhibition via G-quadruplex stabilization is considered a therapeutic strategy against cancer. Theoretically G-quadruplex can form anywhere along the long G-rich strand. Where G-quadruplex forms determines whether the 3' telomere end is accessible to telomerase and may have implications in other functions telomere plays. We investigated G-quadruplex formation at different positions by DMS footprinting and exonuclease hydrolysis. We show that G-quadruplex preferentially forms at the very 3' end than at internal positions. This property provides a molecular basis for telomerase inhibition by G-quadruplex formation. Moreover, it may also regulate those processes that depend on the structure of the very 3' telomere end, for instance, the alternative lengthening of telomere mechanism, telomere T-loop formation, telomere end protection and the replication of bulky telomere DNA. Therefore, targeting telomere G-quadruplex may influence more telomere functions than simply inhibiting telomerase.