INTRODUCTIONTREM2 is an innate immune receptor expressed on myeloid cells including microglia in ... more INTRODUCTIONTREM2 is an innate immune receptor expressed on myeloid cells including microglia in the brain. How TREM2 engages different ligands remains poorly understood.METHODSWe used comprehensive BLI analysis to investigate the TREM2 interactions with ApoE and monomeric amyloid beta (mAβ42).RESULTSTREM2 binding did not depend on ApoE lipidation, and there were only slight differences in affinity observed between ApoE isoforms (E4 > E3 > E2). Surprisingly, disease-linked TREM2 variants within a “basic patch” minimally impact ApoE binding. Instead, TREM2 has a unique hydrophobic surface that can bind to ApoE. This direct engagement requires the hinge region of ApoE. TREM2 directly binds mAβ42 and can potently inhibit Aβ42 polymerization, suggesting a potential mechanism for soluble TREM2 (sTREM2) in preventing AD pathogenesis.DISCUSSIONThese findings demonstrate that TREM2 has at least two separate surfaces to engage ligands and uncovers a potential function for sTREM2 in dir...
Curli amyloid fibers are produced as part of the extracellular biofilm matrix and are composed pr... more Curli amyloid fibers are produced as part of the extracellular biofilm matrix and are composed primarily of the major structural subunit CsgA. The CsgE chaperone facilitates the secretion of CsgA through CsgG by forming a cap at the base of the nonameric CsgG outer membrane pore. We elucidated a series of finely tuned nonpolar and charge-charge interactions that facilitate the oligomerization of CsgE and its ability to transport unfolded CsgA to CsgG for translocation. CsgE oligomerization is temperature dependent and is disrupted by mutations in the W48 and F79 residues. Using nuclear magnetic resonance (NMR), we identified two regions of CsgE involved in the CsgE-CsgA interaction: a head comprising a positively charged patch centered around R47 and a stem comprising a negatively charged patch containing E31 and E85. Negatively charged residues in the intrinsically disordered N- and C-terminal "tails" were not implicated in this interaction. Head and stem residues were mu...
Proceedings of the National Academy of Sciences of the United States of America, Jun 28, 2016
Curli, consisting primarily of major structural subunit CsgA, are functional amyloids produced on... more Curli, consisting primarily of major structural subunit CsgA, are functional amyloids produced on the surface of Escherichia coli, as well as many other enteric bacteria, and are involved in cell colonization and biofilm formation. CsgE is a periplasmic accessory protein that plays a crucial role in curli biogenesis. CsgE binds to both CsgA and the nonameric pore protein CsgG. The CsgG-CsgE complex is the curli secretion channel and is essential for the formation of the curli fibril in vivo. To better understand the role of CsgE in curli formation, we have determined the solution NMR structure of a double mutant of CsgE (W48A/F79A) that appears to be similar to the wild-type (WT) protein in overall structure and function but does not form mixed oligomers at NMR concentrations similar to the WT. The well-converged structure of this mutant has a core scaffold composed of a layer of two α-helices and a layer of three-stranded antiparallel β-sheet with flexible N and C termini. The stru...
NMR studies are now unraveling the structure of intermediates of protein folding using hydrogen—d... more NMR studies are now unraveling the structure of intermediates of protein folding using hydrogen—deuterium exchange methodologies. These studies provide information about the time dependence of formation of secondary structure. They require the ability to assign specific resonances in the NMR spectra to specific amide protons of a protein followed by experiments involving competition between folding and exchange reactions. Another approach is to use 19F‐substituted amino acids to follow changes in side‐chain environment upon folding. Current techniques of molecular biology allow assignments of 19F resonances to specific amino acids by site‐directed mutagenesis. It is possible to follow changes and to analyze results from 19F spectra in real time using a stopped‐flow device incorporated into the NMR spectrometer.
Proceedings of the National Academy of Sciences, 2002
PapD is a periplasmic chaperone essential for P pilus formation in pyelonephritic strains of E. c... more PapD is a periplasmic chaperone essential for P pilus formation in pyelonephritic strains of E. coli . It is composed of two domains, each of which contains a tryptophan residue (Trp-36 and Trp-128, in the N- and C-terminal domains, respectively). To explore the role of domain–domain interactions during folding, the protein was labeled with 6-fluorotryptophan for use in 19 F-NMR experiments. 19 F-NMR data collected as a function of urea concentration revealed the presence of a resonance caused by Trp-128 that was distinct from either the folded or unfolded resonances. The time course of refolding from urea was monitored by stopped-flow fluorescence, CD, and 19 F-NMR, each method showing multiple kinetic phases. The 19 F-NMR stopped-flow spectra, collected at 70 μM of protein with a fluorine cryoprobe, demonstrated that the intermediate was populated early in the folding process (<5 s). The slow disappearance of the intermediate and unfolded resonance occurred at the same rate as ...
Proceedings of the National Academy of Sciences, 2000
A fundamental question in molecular biology is how proteins fold into domains that can serve as a... more A fundamental question in molecular biology is how proteins fold into domains that can serve as assembly modules for building up large macromolecular structures. The biogenesis of pili on the surface of Gram-negative bacteria requires the orchestration of a complex process that includes protein synthesis, folding via small chaperones, secretion, and assembly. The results presented here support the hypothesis that pilus subunit folding and biogenesis proceed via mechanisms termed donor strand complementation and donor strand exchange. Here we show that the steric information necessary for pilus subunit folding is not contained in one polypeptide sequence. Rather, the missing information is transiently donated by a strand of a small chaperone to allow folding. Providing the missing information for folding, via a 13-amino acid peptide extension to the C-terminal end of a pilus subunit, resulted in the production of a protein that no longer required the chaperone to fold. This mechanism...
Proceedings of the National Academy of Sciences, 2006
We have used fluorescence correlation spectroscopy measurements to quantify the hydrodynamic size... more We have used fluorescence correlation spectroscopy measurements to quantify the hydrodynamic sizes of monomeric polyglutamine as a function of chain length ( N ) by measuring the scaling of translational diffusion times (τ D ) for the peptide series (Gly)-(Gln) N -Cys-Lys 2 in aqueous solution. We find that τ D scales with N as τ o N ν and therefore ln(τ D ) = ln(τ o ) + νln( N ). The values for ν and ln(τ o ) are 0.32 ± 0.02 and 3.04 ± 0.08, respectively. Based on these observations, we conclude that water is a polymeric poor solvent for polyglutamine. Previous studies have shown that monomeric polyglutamine is intrinsically disordered. These observations combined with our fluorescence correlation spectroscopy data suggest that the ensemble for monomeric polyglutamine is made up of a heterogeneous collection of collapsed structures. This result is striking because the preference for collapsed structures arises despite the absence of residues deemed to be hydrophobic in the sequence...
Proceedings of the National Academy of Sciences, 1995
Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) contains five tryptophan residues tha... more Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) contains five tryptophan residues that have been replaced with 6-19F-tryptophan. The 19F NMR assignments are known in the native, unliganded form and the unfolded form. We have used these assignments with stopped-flow 19F NMR spectroscopy to investigate the behavior of specific regions of the protein in real time during urea-induced unfolding. The NMR data show that within 1.5 sec most of the intensities of the native 19F resonances of the protein are lost but only a fraction (approximately 20%) of the intensities of the unfolded resonances appears. We postulate that the early disappearance of the native resonances indicates that most of the protein rapidly forms an intermediate in which the side chains have considerable mobility. Stopped-flow far-UV circular dichroism measurements indicate that this intermediate retains native-like secondary structure. Eighty percent of the intensities of the NMR resonances assigned to the...
Proceedings of the National Academy of Sciences, 2012
Curli are extracellular proteinaceous functional amyloid aggregates produced by Escherichia coli ... more Curli are extracellular proteinaceous functional amyloid aggregates produced by Escherichia coli , Salmonella spp., and other enteric bacteria. Curli mediate host cell adhesion and invasion and play a critical role in biofilm formation. Curli filaments consist of CsgA, the major subunit, and CsgB, the minor subunit. In vitro, purified CsgA and CsgB exhibit intrinsically disordered properties, and both are capable of forming amyloid fibers similar in morphology to those formed in vivo. However, in vivo, CsgA alone cannot form curli fibers, and CsgB is required for filament growth. Thus, we studied the aggregation of CsgA and CsgB both alone and together in vitro to investigate the different roles of CsgA and CsgB in curli formation. We found that though CsgA and CsgB individually are able to self-associate to form aggregates/fibrils, they do so using different mechanisms and with different kinetic behavior. CsgB rapidly forms structured oligomers, whereas CsgA aggregation is slower a...
Proceedings of the National Academy of Sciences, 2013
Significance How do N- and C-terminal flanking sequences from exon 1 of the huntingtin protein mo... more Significance How do N- and C-terminal flanking sequences from exon 1 of the huntingtin protein modulate the mechanisms of polyglutamine aggregation? We answer this question using approaches that combine distinct probes of aggregation mechanisms with measurements of solubility and aggregate morphologies. The N- and C-terminal flanking sequence modules from exon 1 of huntingtin act as gatekeepers, whereby the N-terminal flanking sequence accelerates fibril formation while destabilizing nonfibrillar species, whereas the C-terminal flanking sequence reduces the overall driving force for aggregation. These results provide a mechanistic underpinning for observations regarding naturally occurring sequence contexts as modulators of polyglutamine toxicity.
INTRODUCTIONTREM2 is an innate immune receptor expressed on myeloid cells including microglia in ... more INTRODUCTIONTREM2 is an innate immune receptor expressed on myeloid cells including microglia in the brain. How TREM2 engages different ligands remains poorly understood.METHODSWe used comprehensive BLI analysis to investigate the TREM2 interactions with ApoE and monomeric amyloid beta (mAβ42).RESULTSTREM2 binding did not depend on ApoE lipidation, and there were only slight differences in affinity observed between ApoE isoforms (E4 > E3 > E2). Surprisingly, disease-linked TREM2 variants within a “basic patch” minimally impact ApoE binding. Instead, TREM2 has a unique hydrophobic surface that can bind to ApoE. This direct engagement requires the hinge region of ApoE. TREM2 directly binds mAβ42 and can potently inhibit Aβ42 polymerization, suggesting a potential mechanism for soluble TREM2 (sTREM2) in preventing AD pathogenesis.DISCUSSIONThese findings demonstrate that TREM2 has at least two separate surfaces to engage ligands and uncovers a potential function for sTREM2 in dir...
Curli amyloid fibers are produced as part of the extracellular biofilm matrix and are composed pr... more Curli amyloid fibers are produced as part of the extracellular biofilm matrix and are composed primarily of the major structural subunit CsgA. The CsgE chaperone facilitates the secretion of CsgA through CsgG by forming a cap at the base of the nonameric CsgG outer membrane pore. We elucidated a series of finely tuned nonpolar and charge-charge interactions that facilitate the oligomerization of CsgE and its ability to transport unfolded CsgA to CsgG for translocation. CsgE oligomerization is temperature dependent and is disrupted by mutations in the W48 and F79 residues. Using nuclear magnetic resonance (NMR), we identified two regions of CsgE involved in the CsgE-CsgA interaction: a head comprising a positively charged patch centered around R47 and a stem comprising a negatively charged patch containing E31 and E85. Negatively charged residues in the intrinsically disordered N- and C-terminal "tails" were not implicated in this interaction. Head and stem residues were mu...
Proceedings of the National Academy of Sciences of the United States of America, Jun 28, 2016
Curli, consisting primarily of major structural subunit CsgA, are functional amyloids produced on... more Curli, consisting primarily of major structural subunit CsgA, are functional amyloids produced on the surface of Escherichia coli, as well as many other enteric bacteria, and are involved in cell colonization and biofilm formation. CsgE is a periplasmic accessory protein that plays a crucial role in curli biogenesis. CsgE binds to both CsgA and the nonameric pore protein CsgG. The CsgG-CsgE complex is the curli secretion channel and is essential for the formation of the curli fibril in vivo. To better understand the role of CsgE in curli formation, we have determined the solution NMR structure of a double mutant of CsgE (W48A/F79A) that appears to be similar to the wild-type (WT) protein in overall structure and function but does not form mixed oligomers at NMR concentrations similar to the WT. The well-converged structure of this mutant has a core scaffold composed of a layer of two α-helices and a layer of three-stranded antiparallel β-sheet with flexible N and C termini. The stru...
NMR studies are now unraveling the structure of intermediates of protein folding using hydrogen—d... more NMR studies are now unraveling the structure of intermediates of protein folding using hydrogen—deuterium exchange methodologies. These studies provide information about the time dependence of formation of secondary structure. They require the ability to assign specific resonances in the NMR spectra to specific amide protons of a protein followed by experiments involving competition between folding and exchange reactions. Another approach is to use 19F‐substituted amino acids to follow changes in side‐chain environment upon folding. Current techniques of molecular biology allow assignments of 19F resonances to specific amino acids by site‐directed mutagenesis. It is possible to follow changes and to analyze results from 19F spectra in real time using a stopped‐flow device incorporated into the NMR spectrometer.
Proceedings of the National Academy of Sciences, 2002
PapD is a periplasmic chaperone essential for P pilus formation in pyelonephritic strains of E. c... more PapD is a periplasmic chaperone essential for P pilus formation in pyelonephritic strains of E. coli . It is composed of two domains, each of which contains a tryptophan residue (Trp-36 and Trp-128, in the N- and C-terminal domains, respectively). To explore the role of domain–domain interactions during folding, the protein was labeled with 6-fluorotryptophan for use in 19 F-NMR experiments. 19 F-NMR data collected as a function of urea concentration revealed the presence of a resonance caused by Trp-128 that was distinct from either the folded or unfolded resonances. The time course of refolding from urea was monitored by stopped-flow fluorescence, CD, and 19 F-NMR, each method showing multiple kinetic phases. The 19 F-NMR stopped-flow spectra, collected at 70 μM of protein with a fluorine cryoprobe, demonstrated that the intermediate was populated early in the folding process (<5 s). The slow disappearance of the intermediate and unfolded resonance occurred at the same rate as ...
Proceedings of the National Academy of Sciences, 2000
A fundamental question in molecular biology is how proteins fold into domains that can serve as a... more A fundamental question in molecular biology is how proteins fold into domains that can serve as assembly modules for building up large macromolecular structures. The biogenesis of pili on the surface of Gram-negative bacteria requires the orchestration of a complex process that includes protein synthesis, folding via small chaperones, secretion, and assembly. The results presented here support the hypothesis that pilus subunit folding and biogenesis proceed via mechanisms termed donor strand complementation and donor strand exchange. Here we show that the steric information necessary for pilus subunit folding is not contained in one polypeptide sequence. Rather, the missing information is transiently donated by a strand of a small chaperone to allow folding. Providing the missing information for folding, via a 13-amino acid peptide extension to the C-terminal end of a pilus subunit, resulted in the production of a protein that no longer required the chaperone to fold. This mechanism...
Proceedings of the National Academy of Sciences, 2006
We have used fluorescence correlation spectroscopy measurements to quantify the hydrodynamic size... more We have used fluorescence correlation spectroscopy measurements to quantify the hydrodynamic sizes of monomeric polyglutamine as a function of chain length ( N ) by measuring the scaling of translational diffusion times (τ D ) for the peptide series (Gly)-(Gln) N -Cys-Lys 2 in aqueous solution. We find that τ D scales with N as τ o N ν and therefore ln(τ D ) = ln(τ o ) + νln( N ). The values for ν and ln(τ o ) are 0.32 ± 0.02 and 3.04 ± 0.08, respectively. Based on these observations, we conclude that water is a polymeric poor solvent for polyglutamine. Previous studies have shown that monomeric polyglutamine is intrinsically disordered. These observations combined with our fluorescence correlation spectroscopy data suggest that the ensemble for monomeric polyglutamine is made up of a heterogeneous collection of collapsed structures. This result is striking because the preference for collapsed structures arises despite the absence of residues deemed to be hydrophobic in the sequence...
Proceedings of the National Academy of Sciences, 1995
Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) contains five tryptophan residues tha... more Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) contains five tryptophan residues that have been replaced with 6-19F-tryptophan. The 19F NMR assignments are known in the native, unliganded form and the unfolded form. We have used these assignments with stopped-flow 19F NMR spectroscopy to investigate the behavior of specific regions of the protein in real time during urea-induced unfolding. The NMR data show that within 1.5 sec most of the intensities of the native 19F resonances of the protein are lost but only a fraction (approximately 20%) of the intensities of the unfolded resonances appears. We postulate that the early disappearance of the native resonances indicates that most of the protein rapidly forms an intermediate in which the side chains have considerable mobility. Stopped-flow far-UV circular dichroism measurements indicate that this intermediate retains native-like secondary structure. Eighty percent of the intensities of the NMR resonances assigned to the...
Proceedings of the National Academy of Sciences, 2012
Curli are extracellular proteinaceous functional amyloid aggregates produced by Escherichia coli ... more Curli are extracellular proteinaceous functional amyloid aggregates produced by Escherichia coli , Salmonella spp., and other enteric bacteria. Curli mediate host cell adhesion and invasion and play a critical role in biofilm formation. Curli filaments consist of CsgA, the major subunit, and CsgB, the minor subunit. In vitro, purified CsgA and CsgB exhibit intrinsically disordered properties, and both are capable of forming amyloid fibers similar in morphology to those formed in vivo. However, in vivo, CsgA alone cannot form curli fibers, and CsgB is required for filament growth. Thus, we studied the aggregation of CsgA and CsgB both alone and together in vitro to investigate the different roles of CsgA and CsgB in curli formation. We found that though CsgA and CsgB individually are able to self-associate to form aggregates/fibrils, they do so using different mechanisms and with different kinetic behavior. CsgB rapidly forms structured oligomers, whereas CsgA aggregation is slower a...
Proceedings of the National Academy of Sciences, 2013
Significance How do N- and C-terminal flanking sequences from exon 1 of the huntingtin protein mo... more Significance How do N- and C-terminal flanking sequences from exon 1 of the huntingtin protein modulate the mechanisms of polyglutamine aggregation? We answer this question using approaches that combine distinct probes of aggregation mechanisms with measurements of solubility and aggregate morphologies. The N- and C-terminal flanking sequence modules from exon 1 of huntingtin act as gatekeepers, whereby the N-terminal flanking sequence accelerates fibril formation while destabilizing nonfibrillar species, whereas the C-terminal flanking sequence reduces the overall driving force for aggregation. These results provide a mechanistic underpinning for observations regarding naturally occurring sequence contexts as modulators of polyglutamine toxicity.
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