Oxidized cysteine residues are highly reactive and can form functional covalent conjugates, of wh... more Oxidized cysteine residues are highly reactive and can form functional covalent conjugates, of which the allosteric redox switch formed by the lysine-cysteine NOS bridge is an example. Here, we report a noncanonical FAD-dependent enzyme Orf1 that adds a glycine-derived N-formimidoyl group to glycinothricin to form the antibiotic BD-12. X-ray crystallography was used to investigate this complex enzymatic process, which showed Orf1 has two substrate-binding sites that sit 13.5 Å apart unlike canonical FAD-dependent oxidoreductases. One site could accommodate glycine and the other glycinothricin or glycylthricin. Moreover, an intermediate-enzyme adduct with a NOS-covalent linkage was observed in the later site, where it acts as a two-scissile-bond linkage facilitating nucleophilic addition and cofactor-free decarboxylation. The chain length of nucleophilic acceptors vies with bond cleavage sites at either N–O or O–S accounting for N-formimidoylation or N-iminoacetylation. The resultant...
SARS-CoV-2 is a novel pathogen causing pneumonia named COVID-19 and leading to a severe pandemic ... more SARS-CoV-2 is a novel pathogen causing pneumonia named COVID-19 and leading to a severe pandemic since the end of 2019. The genome of SARS-CoV-2 contains a macro domain that may play an important role in regulating ADP-ribosylation in host cells and initiating viral replication. Here, we report the 1 H, 13 C, and 15 N resonance assignments of the SARS-CoV-2 macro domain. This work provides the ground for further structural deciphering and biophysical investigation in protein function and antiviral agent design. Keywords COVID-19 • SARS-CoV-2 • Macro domain • Viral protein Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
4-Mercapto-4-methyl-2-pentanone (4MMP), a high-impact aroma compound with the box tree and black ... more 4-Mercapto-4-methyl-2-pentanone (4MMP), a high-impact aroma compound with the box tree and black currant flavors was first identified in wines and could be released by microbial cysteine-S-conjugate β-lyases from its precursors. In this study, various yeasts and bacteria encoding β-lyases were selected to examine their β-lyase activities. A thiol precursor of 4MMP, cysteine-conjugate of 4MMP (cys-4MMP), was synthesized with a purity of >95% in a relatively environmentally friendly approach, and its chemical structure was confirmed by nuclear magnetic resonance spectroscopy. The β-lyase activities of the crude cell extract from the bacteria and yeast strains for different substrates were examined using a colorimetric method. Shewanella putrefaciens cell extract exhibited the highest β-lyase activity for all tested substrates. Additionally, the optimum pH and temperature for their β-lyase activities were determined. To monitor the conversion efficiency of precursor cys-4MMP to 4MMP...
Inhibitor-1 is converted into a potent inhibitor of native protein phosphatase-1 (PP1) when Thr35... more Inhibitor-1 is converted into a potent inhibitor of native protein phosphatase-1 (PP1) when Thr35 is phosphorylated by cAMP-dependent protein kinase (PKA). However, PKA-phosphorylated form of inhibitor-1 displayed a weak activity in inhibition of recombinant PP1. The mechanism for the impaired activity of PKA-phosphorylated inhibitor-1 toward inhibition of recombinant PP1 remained elusive. By using NMR spectroscopy in combination with site-directed mutagenesis and inhibitory assay, we found that the interaction between recombinant PP1 and the consensus PP1-binding motif of PKA-thiophosphorylated form of inhibitor-1 was unexpectedly weak. Unlike binding to native PP1, the subdomains 1 (residues around and including the phosphorylated Thr35) and 2 (the consensus PP1-binding motif) of PKA-thiophosphorylated form of inhibitor-1 do not exhibit a synergistic effect in inhibition of recombinant PP1. This finding implied that a slight structural discrepancy exists between native and recombi...
Alzheimer's disease (AD) is the most common dementia affecting tens of million people worldwi... more Alzheimer's disease (AD) is the most common dementia affecting tens of million people worldwide. The primary neuropathological hallmark in AD is amyloid plaques composed of amyloid-β peptide (Aβ). Several familial mutations found in Aβ sequence result in early onset of AD. Previous studies showed that the mutations located at N-terminus of Aβ, such as the English (H6R) and Tottori (D7N) mutations, promote fibril formation and increase cytotoxicity. However, A2T mutant located at the very N-terminus of Aβ shows low-prevalence incidence of AD, whereas, another mutant A2V causes early onset of AD. To understand the molecular mechanism of the distinct effect and develop new potential therapeutic strategy, here, we examined the effect of full-length and N-terminal A2V/T variants to wild type (WT) Aβ40 by fibrillization assays and NMR studies. We found that full-length and N-terminal A2V accelerated WT fibrillization and induced large chemical shifts on the N-terminus of WT Aβ, wherea...
β-amyloid peptide (Aβ) aggregation has been thought to be associated with the pathogenesis of Alz... more β-amyloid peptide (Aβ) aggregation has been thought to be associated with the pathogenesis of Alzheimer's disease. Recently, we showed that L17A/F19A substitutions may increase the structural stability of wild-type and Arctic-type Aβ 40 and decrease the rates of structural conversion and fibril formation. However, the underlying mechanism for the increase of structural stability as a result of the alanine substitutions remained elusive. In this study, we apply nuclear magnetic resonance and circular dichroism spectroscopies to characterize the Aβ 40 structure, demonstrating that L17A/F19A substitutions can augment the α-helicity of the residues located in the α/β-discordant segment (resides 15 to 23) of both wild-type and Arctic-type Aβ 40. These results provide a structural basis to link the αhelicity of the α/β-discordant segment with the conformational conversion propensity of Aβ.
The newly emerging human pathogen, Middle East respiratory syndrome coronavirus (MERS-CoV), conta... more The newly emerging human pathogen, Middle East respiratory syndrome coronavirus (MERS-CoV), contains a macro domain in the highly conserved N-terminal region of non-structural protein 3. Intense research has shown that macro domains bind ADP-ribose and other derivatives, but it still remains intangible about their exact function. In this study we report the preliminary structural analysis through solution NMR spectroscopy of the MERS-CoV macro domain. The near complete NMR assignments of MERS-CoV macro domain provide the basis for subsequent structural and biochemical investigation in the context of protein function. Keywords Viral macro domain Á MERS-CoV Á ADPribose binding module Á NMR spectroscopy Biological context Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a kind of RNA virus belongs to genus Betacoronavirus in Coronavaridae of enveloped, which is a positive-stranded RNA virus and becomes a novel zoonotic virus in recent years (de Groot et al. 2013). MERS-CoV transmits respiratory tract infections from original host, the bat, to human by intermediate host, the camel, although the detail viral life cycle is still unknown (Han et al. 2015). The first human case was reported in 2012 at Saudi Arabia about a man with severe acute pneumonia and secondary renal failure (Zaki et al. 2012). Through close contact to infected patients, the human-to-human transmissions of the MERS-CoV virus spread very quickly. Since the first report, MERS-CoV has caused over 400 people died among 1100 cases in 23 countries. There is no efficient specific drug to use for this newly human pathogen yet, thus drug development is the emergency to conquer for the researchers. The macro domain, a protein module specifically recognizing ADP ribose (Karras et al. 2005), could be found in many bacteria, archaea, plants and vertebrates, suggesting their evolutionary conservation (Han et al. 2011). Some macro domains possess phosphatase activity towards ADP-ribose-1 00-phosphate, which is a by-product of tRNA splicing (Putic et al. 2005). Furthermore, macro domains are found in nonstructural proteins (nsps) of several positive-strand RNA viruses, including alphaviruses, rubella virus, hepatitis E virus (HEV), and coronaviruses [including the severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV)] (Neuvonen and Ahola 2009; Fehr and Perlman 2015; Hurst-Hess et al. 2015; Parvez 2015). The enzymatic activity of viral macro domain is Yi-Ping Huang and Chao-Cheng Cho have contributed equally to this work.
The RstA/RstB system is a bacterial two-component regulatory system consisting of the membrane se... more The RstA/RstB system is a bacterial two-component regulatory system consisting of the membrane sensor, RstB and its cognate response regulator (RR) RstA. The RstA of Klebsiella pneumoniae (kpRstA) consists of an N-terminal receiver domain (RD, residues 1-119) and a C-terminal DNA-binding domain (DBD, residues 130-236). Phosphorylation of kpRstA induces dimerization, which allows two kpRstA DBDs to bind to a tandem repeat, called the RstA box, and regulate the expression of downstream genes. Here we report the solution and crystal structures of the free kpRstA RD, DBD and DBD/RstA box DNA complex. The structure of the kpRstA DBD/RstA box complex suggests that the two protomers interact with the RstA box in an asymmetric fashion. Equilibrium binding studies further reveal that the two protomers within the kpRstA dimer bind to the RstA box in a sequential manner. Taken together, our results suggest a binding model where dimerization of the kpRstA RDs provides the platform to allow the ...
Full wwPDB NMR Structure Validation Report 2MWX 2 Ensemble composition and analysis i O This entr... more Full wwPDB NMR Structure Validation Report 2MWX 2 Ensemble composition and analysis i O This entry contains 20 models. Model 20 is the overall representative, medoid model (most similar to other models). The authors have identied model 1 as representative, based on the following criterion: lowest energy. The following residues are included in the computation of the global validation metrics. Well-dened (core) protein residues Well-dened core Residue range (total) Backbone RMSD (Å) Medoid model
The E3 ubiquitin ligase RNF4 (RING finger protein 4) contains four tandem SIM [SUMO (small ubiqui... more The E3 ubiquitin ligase RNF4 (RING finger protein 4) contains four tandem SIM [SUMO (small ubiquitin-like modifier)-interaction motif] repeats for selective interaction with poly-SUMO-modified proteins, which it targets for degradation. We employed a multi-faceted approach to characterize the structure of the RNF4-SIMs domain and the tetra-SUMO2 chain to elucidate the interaction between them. In solution, the SIM domain was intrinsically disordered and the linkers of the tetra-SUMO2 were highly flexible. Individual SIMs of the RNF4-SIMs domains bind to SUMO2 in the groove between the β2-strand and the α1-helix parallel to the β2-strand. SIM2 and SIM3 bound to SUMO with a high affinity and together constituted the recognition module necessary for SUMO binding. SIM4 alone bound to SUMO with low affinity; however, its contribution to tetra-SUMO2 binding avidity is comparable with that of SIM3 when in the RNF4-SIMs domain. The SAXS data of the tetra-SUMO2-RNF4-SIMs domain complex indic...
Small cystine-stabilized proteins are desirable scaffolds for therapeutics and diagnostics. Speci... more Small cystine-stabilized proteins are desirable scaffolds for therapeutics and diagnostics. Specific folding and binding properties of the proteinaceous binders can be engineered with combinatorial protein libraries in connection with artificial molecular evolution. The combinatorial protein libraries are composed of scaffold variants with random sequence variation, which inevitably produces a portion of the library sequences incompatible with the parent structure. Here, we used artificial molecular evolution to elucidate structure-determining residues in a smallest cystine-stabilized scaffold. The structural determinant information was then applied to designing cystine-stabilized miniproteins binding to human vascular endothelial growth factor. This work demonstrated a general methodology on engineering artificial cystine-stabilized proteins as antibody mimetics with simultaneously enhanced folding and binding properties.
Blo t 5 is the major allergen from Blomia tropicalis mites and shows strong IgE reactivity with u... more Blo t 5 is the major allergen from Blomia tropicalis mites and shows strong IgE reactivity with up to 90% of asthmatic and rhinitis patients' sera. The NMR solution structure of Blo t 5 comprises three long a helices, forming a coiled-coil, triple-helical bundle with a left-handed twist. TROSY-NMR experiments were used to study Blo t 5 interaction with the Fab 0 of a specific monoclonal antibody, mAb 4A7. The mAb epitope comprises two closely spaced surfaces, I and II, connected by a sharp turn and bearing critical residues Asn46 and Lys47 on one surface, and Lys54 and Arg57 on the other. This discontinuous epitope overlaps with the human IgE epitope(s) of Blo t 5. Epitope surface II is further predicted to undergo conformational exchange in the millisecond to microsecond range. The results presented are critical for the design of a hypoallergenic Blo t 5 for efficacious immunotherapy of allergic diseases.
NLRPs (Nucleotide-binding domain, leucine-rich repeat and pyrin domain containing proteins) are a... more NLRPs (Nucleotide-binding domain, leucine-rich repeat and pyrin domain containing proteins) are a family of patternrecognition receptors (PRRs) that sense intracellular microbial components and endogenous stress signals. NLRP10 (also known as PYNOD) is a unique NLRP member characterized by a lack of the putative ligand-binding leucine-rich repeat domain. Recently, human NLRP10 has been shown to inhibit the self-association of ASC into aggregates and ASC-mediated procaspase-1 processing. However, such activities are not found in mouse NLRP10. Here we report the solution structure and dynamics of human NLRP10 pyrin domain (PYD), whose helix H3 and loop H2-H3 adopt a conformation distinct from those of mouse NLRP10. Docking studies show that human and mouse NLRP10 PYDs may interact differently with ASC PYD. These results provide a possible structural explanation for the contrasting effect of NLRP10 on ASC aggregation in human cells versus mouse models. Finally, we also provide evidence that in human NLRP10 the PYD domain may not interact with the NOD domain to regulate its intrinsic nucleotide hydrolysis activity.
Amyloid fibrils of a-synuclein are the main constituent of Lewy bodies deposited in substantial n... more Amyloid fibrils of a-synuclein are the main constituent of Lewy bodies deposited in substantial nigra of Parkinson's disease brains. a-Synuclein is an intrinsically disordered protein lacking compact secondary and tertiary structures. To enhance the understanding of its structure and function relationship, we utilized temperature treatment to study a-synuclein conformational changes and the subsequent effects. We found that after 1 hr of high temperature pretreatment, .80uC, asynuclein fibrillization was significantly inhibited. However, the temperature melting coupled with circular dichroism spectra showed that a-synuclein was fully reversible and the NMR studies showed no observable structural changes of a-synuclein after 95uC treatment. By using cross-linking and analytical ultracentrifugation, rare amount of pre-existing a-synuclein oligomers were found to decrease after the high temperature treatment. In addition, a small portion of C-terminal truncation of a-synuclein also occurred. The reduction of pre-existing oligomers of a-synuclein may contribute to less seeding effect that retards the kinetics of amyloid fibrillization. Overall, our results showed that the pre-existing oligomeric species is a key factor contributing to a-synuclein fibrillization. Our results facilitate the understanding of a-synuclein fibrillization.
Iron-inducible transcription of the ap65-1 gene in Trichomonas vaginalis involves at least three ... more Iron-inducible transcription of the ap65-1 gene in Trichomonas vaginalis involves at least three Myb-like transcriptional factors (tvMyb1, tvMyb2 and tvMyb3) that differentially bind to two closely spaced promoter sites, MRE-1/MRE-2r and MRE-2f. Here, we defined a fragment of tvMyb2 comprising residues 40-156 (tvMyb2 40-156) as the minimum structural unit that retains near full binding affinity with the promoter DNAs. Like c-Myb in vertebrates, the DNA-free tvMyb2 40-156 has a flexible and open conformation. Upon binding to the promoter DNA elements, tvMyb2 40-156 undergoes significant conformational rearrangement and structure stabilization. Crystal structures of tvMyb2 40-156 in complex with promoter element-containing DNA oligomers showed that 5 0-a/gACGAT-3 0 is the specific base sequence recognized by tvMyb2 40-156 , which does not fully conform to that of the Myb binding site sequence. Furthermore, Lys 49 , which is upstream of the R2 motif (amino acids 52-102) also participates in specific DNA sequence recognition. Intriguingly, tvMyb2 40-156 binds to the promoter elements in an orientation opposite to that proposed in the HADDOCK model of the tvMyb1 35-141 /MRE-1-MRE-2r complex. These results shed new light on understanding the molecular mechanism of Myb-DNA recognition and provide a framework to study the molecular basis of transcriptional regulation of myriad Mybs in T. vaginalis.
Figure 2. Stereoview of detailed binding interactions of FHA1-pT(Mdt1) (a) in comparisons with th... more Figure 2. Stereoview of detailed binding interactions of FHA1-pT(Mdt1) (a) in comparisons with those of FHA1-pT(Rad9) (b). The ionic interactions involving phosphate group, hydrophobic interactions, and comparable electrostatic/amino-aromatic interactions involving R83 are indicated in red, black, and green, respectively.
The lipoyl-bearing domain (LBD) of the transacylase (E2) subunit of the branched-chain ␣-keto aci... more The lipoyl-bearing domain (LBD) of the transacylase (E2) subunit of the branched-chain ␣-keto acid dehydrogenase complex plays a central role in substrate channeling in this mitochondrial multienzyme complex. We have employed multidimensional heteronuclear NMR techniques to determine the structure and dynamics of the LBD of the human branched-chain ␣-keto acid dehydrogenase complex (hbLBD). Similar to LBD from other members of the ␣-keto acid dehydrogenase family, the solution structure of hbLBD is a flattened -barrel formed by two four-stranded antiparallel -sheets. The lipoyl Lys 44 residue resides at the tip of a -hairpin comprising a sharp type I -turn and the two connecting -strands 4 and 5. A prominent V-shaped groove formed by a surface loop, L1, connecting 1and 2-strands and the lipoyl lysine -hairpin constitutes the functional pocket. We further applied reduced spectral density functions formalism to extract dynamic information of hbLBD from 15 N-T 1 , 15 N-T 2 , and (1 H-15 N) nuclear Overhauser effect data obtained at 600 MHz. The results showed that residues surrounding the lipoyl lysine region comprising the L1 loop and the Lys 44 -turn are highly flexible, whereas -sheet S1 appears to display a slow conformational exchange process.
Oxidized cysteine residues are highly reactive and can form functional covalent conjugates, of wh... more Oxidized cysteine residues are highly reactive and can form functional covalent conjugates, of which the allosteric redox switch formed by the lysine-cysteine NOS bridge is an example. Here, we report a noncanonical FAD-dependent enzyme Orf1 that adds a glycine-derived N-formimidoyl group to glycinothricin to form the antibiotic BD-12. X-ray crystallography was used to investigate this complex enzymatic process, which showed Orf1 has two substrate-binding sites that sit 13.5 Å apart unlike canonical FAD-dependent oxidoreductases. One site could accommodate glycine and the other glycinothricin or glycylthricin. Moreover, an intermediate-enzyme adduct with a NOS-covalent linkage was observed in the later site, where it acts as a two-scissile-bond linkage facilitating nucleophilic addition and cofactor-free decarboxylation. The chain length of nucleophilic acceptors vies with bond cleavage sites at either N–O or O–S accounting for N-formimidoylation or N-iminoacetylation. The resultant...
SARS-CoV-2 is a novel pathogen causing pneumonia named COVID-19 and leading to a severe pandemic ... more SARS-CoV-2 is a novel pathogen causing pneumonia named COVID-19 and leading to a severe pandemic since the end of 2019. The genome of SARS-CoV-2 contains a macro domain that may play an important role in regulating ADP-ribosylation in host cells and initiating viral replication. Here, we report the 1 H, 13 C, and 15 N resonance assignments of the SARS-CoV-2 macro domain. This work provides the ground for further structural deciphering and biophysical investigation in protein function and antiviral agent design. Keywords COVID-19 • SARS-CoV-2 • Macro domain • Viral protein Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
4-Mercapto-4-methyl-2-pentanone (4MMP), a high-impact aroma compound with the box tree and black ... more 4-Mercapto-4-methyl-2-pentanone (4MMP), a high-impact aroma compound with the box tree and black currant flavors was first identified in wines and could be released by microbial cysteine-S-conjugate β-lyases from its precursors. In this study, various yeasts and bacteria encoding β-lyases were selected to examine their β-lyase activities. A thiol precursor of 4MMP, cysteine-conjugate of 4MMP (cys-4MMP), was synthesized with a purity of >95% in a relatively environmentally friendly approach, and its chemical structure was confirmed by nuclear magnetic resonance spectroscopy. The β-lyase activities of the crude cell extract from the bacteria and yeast strains for different substrates were examined using a colorimetric method. Shewanella putrefaciens cell extract exhibited the highest β-lyase activity for all tested substrates. Additionally, the optimum pH and temperature for their β-lyase activities were determined. To monitor the conversion efficiency of precursor cys-4MMP to 4MMP...
Inhibitor-1 is converted into a potent inhibitor of native protein phosphatase-1 (PP1) when Thr35... more Inhibitor-1 is converted into a potent inhibitor of native protein phosphatase-1 (PP1) when Thr35 is phosphorylated by cAMP-dependent protein kinase (PKA). However, PKA-phosphorylated form of inhibitor-1 displayed a weak activity in inhibition of recombinant PP1. The mechanism for the impaired activity of PKA-phosphorylated inhibitor-1 toward inhibition of recombinant PP1 remained elusive. By using NMR spectroscopy in combination with site-directed mutagenesis and inhibitory assay, we found that the interaction between recombinant PP1 and the consensus PP1-binding motif of PKA-thiophosphorylated form of inhibitor-1 was unexpectedly weak. Unlike binding to native PP1, the subdomains 1 (residues around and including the phosphorylated Thr35) and 2 (the consensus PP1-binding motif) of PKA-thiophosphorylated form of inhibitor-1 do not exhibit a synergistic effect in inhibition of recombinant PP1. This finding implied that a slight structural discrepancy exists between native and recombi...
Alzheimer's disease (AD) is the most common dementia affecting tens of million people worldwi... more Alzheimer's disease (AD) is the most common dementia affecting tens of million people worldwide. The primary neuropathological hallmark in AD is amyloid plaques composed of amyloid-β peptide (Aβ). Several familial mutations found in Aβ sequence result in early onset of AD. Previous studies showed that the mutations located at N-terminus of Aβ, such as the English (H6R) and Tottori (D7N) mutations, promote fibril formation and increase cytotoxicity. However, A2T mutant located at the very N-terminus of Aβ shows low-prevalence incidence of AD, whereas, another mutant A2V causes early onset of AD. To understand the molecular mechanism of the distinct effect and develop new potential therapeutic strategy, here, we examined the effect of full-length and N-terminal A2V/T variants to wild type (WT) Aβ40 by fibrillization assays and NMR studies. We found that full-length and N-terminal A2V accelerated WT fibrillization and induced large chemical shifts on the N-terminus of WT Aβ, wherea...
β-amyloid peptide (Aβ) aggregation has been thought to be associated with the pathogenesis of Alz... more β-amyloid peptide (Aβ) aggregation has been thought to be associated with the pathogenesis of Alzheimer's disease. Recently, we showed that L17A/F19A substitutions may increase the structural stability of wild-type and Arctic-type Aβ 40 and decrease the rates of structural conversion and fibril formation. However, the underlying mechanism for the increase of structural stability as a result of the alanine substitutions remained elusive. In this study, we apply nuclear magnetic resonance and circular dichroism spectroscopies to characterize the Aβ 40 structure, demonstrating that L17A/F19A substitutions can augment the α-helicity of the residues located in the α/β-discordant segment (resides 15 to 23) of both wild-type and Arctic-type Aβ 40. These results provide a structural basis to link the αhelicity of the α/β-discordant segment with the conformational conversion propensity of Aβ.
The newly emerging human pathogen, Middle East respiratory syndrome coronavirus (MERS-CoV), conta... more The newly emerging human pathogen, Middle East respiratory syndrome coronavirus (MERS-CoV), contains a macro domain in the highly conserved N-terminal region of non-structural protein 3. Intense research has shown that macro domains bind ADP-ribose and other derivatives, but it still remains intangible about their exact function. In this study we report the preliminary structural analysis through solution NMR spectroscopy of the MERS-CoV macro domain. The near complete NMR assignments of MERS-CoV macro domain provide the basis for subsequent structural and biochemical investigation in the context of protein function. Keywords Viral macro domain Á MERS-CoV Á ADPribose binding module Á NMR spectroscopy Biological context Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a kind of RNA virus belongs to genus Betacoronavirus in Coronavaridae of enveloped, which is a positive-stranded RNA virus and becomes a novel zoonotic virus in recent years (de Groot et al. 2013). MERS-CoV transmits respiratory tract infections from original host, the bat, to human by intermediate host, the camel, although the detail viral life cycle is still unknown (Han et al. 2015). The first human case was reported in 2012 at Saudi Arabia about a man with severe acute pneumonia and secondary renal failure (Zaki et al. 2012). Through close contact to infected patients, the human-to-human transmissions of the MERS-CoV virus spread very quickly. Since the first report, MERS-CoV has caused over 400 people died among 1100 cases in 23 countries. There is no efficient specific drug to use for this newly human pathogen yet, thus drug development is the emergency to conquer for the researchers. The macro domain, a protein module specifically recognizing ADP ribose (Karras et al. 2005), could be found in many bacteria, archaea, plants and vertebrates, suggesting their evolutionary conservation (Han et al. 2011). Some macro domains possess phosphatase activity towards ADP-ribose-1 00-phosphate, which is a by-product of tRNA splicing (Putic et al. 2005). Furthermore, macro domains are found in nonstructural proteins (nsps) of several positive-strand RNA viruses, including alphaviruses, rubella virus, hepatitis E virus (HEV), and coronaviruses [including the severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV)] (Neuvonen and Ahola 2009; Fehr and Perlman 2015; Hurst-Hess et al. 2015; Parvez 2015). The enzymatic activity of viral macro domain is Yi-Ping Huang and Chao-Cheng Cho have contributed equally to this work.
The RstA/RstB system is a bacterial two-component regulatory system consisting of the membrane se... more The RstA/RstB system is a bacterial two-component regulatory system consisting of the membrane sensor, RstB and its cognate response regulator (RR) RstA. The RstA of Klebsiella pneumoniae (kpRstA) consists of an N-terminal receiver domain (RD, residues 1-119) and a C-terminal DNA-binding domain (DBD, residues 130-236). Phosphorylation of kpRstA induces dimerization, which allows two kpRstA DBDs to bind to a tandem repeat, called the RstA box, and regulate the expression of downstream genes. Here we report the solution and crystal structures of the free kpRstA RD, DBD and DBD/RstA box DNA complex. The structure of the kpRstA DBD/RstA box complex suggests that the two protomers interact with the RstA box in an asymmetric fashion. Equilibrium binding studies further reveal that the two protomers within the kpRstA dimer bind to the RstA box in a sequential manner. Taken together, our results suggest a binding model where dimerization of the kpRstA RDs provides the platform to allow the ...
Full wwPDB NMR Structure Validation Report 2MWX 2 Ensemble composition and analysis i O This entr... more Full wwPDB NMR Structure Validation Report 2MWX 2 Ensemble composition and analysis i O This entry contains 20 models. Model 20 is the overall representative, medoid model (most similar to other models). The authors have identied model 1 as representative, based on the following criterion: lowest energy. The following residues are included in the computation of the global validation metrics. Well-dened (core) protein residues Well-dened core Residue range (total) Backbone RMSD (Å) Medoid model
The E3 ubiquitin ligase RNF4 (RING finger protein 4) contains four tandem SIM [SUMO (small ubiqui... more The E3 ubiquitin ligase RNF4 (RING finger protein 4) contains four tandem SIM [SUMO (small ubiquitin-like modifier)-interaction motif] repeats for selective interaction with poly-SUMO-modified proteins, which it targets for degradation. We employed a multi-faceted approach to characterize the structure of the RNF4-SIMs domain and the tetra-SUMO2 chain to elucidate the interaction between them. In solution, the SIM domain was intrinsically disordered and the linkers of the tetra-SUMO2 were highly flexible. Individual SIMs of the RNF4-SIMs domains bind to SUMO2 in the groove between the β2-strand and the α1-helix parallel to the β2-strand. SIM2 and SIM3 bound to SUMO with a high affinity and together constituted the recognition module necessary for SUMO binding. SIM4 alone bound to SUMO with low affinity; however, its contribution to tetra-SUMO2 binding avidity is comparable with that of SIM3 when in the RNF4-SIMs domain. The SAXS data of the tetra-SUMO2-RNF4-SIMs domain complex indic...
Small cystine-stabilized proteins are desirable scaffolds for therapeutics and diagnostics. Speci... more Small cystine-stabilized proteins are desirable scaffolds for therapeutics and diagnostics. Specific folding and binding properties of the proteinaceous binders can be engineered with combinatorial protein libraries in connection with artificial molecular evolution. The combinatorial protein libraries are composed of scaffold variants with random sequence variation, which inevitably produces a portion of the library sequences incompatible with the parent structure. Here, we used artificial molecular evolution to elucidate structure-determining residues in a smallest cystine-stabilized scaffold. The structural determinant information was then applied to designing cystine-stabilized miniproteins binding to human vascular endothelial growth factor. This work demonstrated a general methodology on engineering artificial cystine-stabilized proteins as antibody mimetics with simultaneously enhanced folding and binding properties.
Blo t 5 is the major allergen from Blomia tropicalis mites and shows strong IgE reactivity with u... more Blo t 5 is the major allergen from Blomia tropicalis mites and shows strong IgE reactivity with up to 90% of asthmatic and rhinitis patients' sera. The NMR solution structure of Blo t 5 comprises three long a helices, forming a coiled-coil, triple-helical bundle with a left-handed twist. TROSY-NMR experiments were used to study Blo t 5 interaction with the Fab 0 of a specific monoclonal antibody, mAb 4A7. The mAb epitope comprises two closely spaced surfaces, I and II, connected by a sharp turn and bearing critical residues Asn46 and Lys47 on one surface, and Lys54 and Arg57 on the other. This discontinuous epitope overlaps with the human IgE epitope(s) of Blo t 5. Epitope surface II is further predicted to undergo conformational exchange in the millisecond to microsecond range. The results presented are critical for the design of a hypoallergenic Blo t 5 for efficacious immunotherapy of allergic diseases.
NLRPs (Nucleotide-binding domain, leucine-rich repeat and pyrin domain containing proteins) are a... more NLRPs (Nucleotide-binding domain, leucine-rich repeat and pyrin domain containing proteins) are a family of patternrecognition receptors (PRRs) that sense intracellular microbial components and endogenous stress signals. NLRP10 (also known as PYNOD) is a unique NLRP member characterized by a lack of the putative ligand-binding leucine-rich repeat domain. Recently, human NLRP10 has been shown to inhibit the self-association of ASC into aggregates and ASC-mediated procaspase-1 processing. However, such activities are not found in mouse NLRP10. Here we report the solution structure and dynamics of human NLRP10 pyrin domain (PYD), whose helix H3 and loop H2-H3 adopt a conformation distinct from those of mouse NLRP10. Docking studies show that human and mouse NLRP10 PYDs may interact differently with ASC PYD. These results provide a possible structural explanation for the contrasting effect of NLRP10 on ASC aggregation in human cells versus mouse models. Finally, we also provide evidence that in human NLRP10 the PYD domain may not interact with the NOD domain to regulate its intrinsic nucleotide hydrolysis activity.
Amyloid fibrils of a-synuclein are the main constituent of Lewy bodies deposited in substantial n... more Amyloid fibrils of a-synuclein are the main constituent of Lewy bodies deposited in substantial nigra of Parkinson's disease brains. a-Synuclein is an intrinsically disordered protein lacking compact secondary and tertiary structures. To enhance the understanding of its structure and function relationship, we utilized temperature treatment to study a-synuclein conformational changes and the subsequent effects. We found that after 1 hr of high temperature pretreatment, .80uC, asynuclein fibrillization was significantly inhibited. However, the temperature melting coupled with circular dichroism spectra showed that a-synuclein was fully reversible and the NMR studies showed no observable structural changes of a-synuclein after 95uC treatment. By using cross-linking and analytical ultracentrifugation, rare amount of pre-existing a-synuclein oligomers were found to decrease after the high temperature treatment. In addition, a small portion of C-terminal truncation of a-synuclein also occurred. The reduction of pre-existing oligomers of a-synuclein may contribute to less seeding effect that retards the kinetics of amyloid fibrillization. Overall, our results showed that the pre-existing oligomeric species is a key factor contributing to a-synuclein fibrillization. Our results facilitate the understanding of a-synuclein fibrillization.
Iron-inducible transcription of the ap65-1 gene in Trichomonas vaginalis involves at least three ... more Iron-inducible transcription of the ap65-1 gene in Trichomonas vaginalis involves at least three Myb-like transcriptional factors (tvMyb1, tvMyb2 and tvMyb3) that differentially bind to two closely spaced promoter sites, MRE-1/MRE-2r and MRE-2f. Here, we defined a fragment of tvMyb2 comprising residues 40-156 (tvMyb2 40-156) as the minimum structural unit that retains near full binding affinity with the promoter DNAs. Like c-Myb in vertebrates, the DNA-free tvMyb2 40-156 has a flexible and open conformation. Upon binding to the promoter DNA elements, tvMyb2 40-156 undergoes significant conformational rearrangement and structure stabilization. Crystal structures of tvMyb2 40-156 in complex with promoter element-containing DNA oligomers showed that 5 0-a/gACGAT-3 0 is the specific base sequence recognized by tvMyb2 40-156 , which does not fully conform to that of the Myb binding site sequence. Furthermore, Lys 49 , which is upstream of the R2 motif (amino acids 52-102) also participates in specific DNA sequence recognition. Intriguingly, tvMyb2 40-156 binds to the promoter elements in an orientation opposite to that proposed in the HADDOCK model of the tvMyb1 35-141 /MRE-1-MRE-2r complex. These results shed new light on understanding the molecular mechanism of Myb-DNA recognition and provide a framework to study the molecular basis of transcriptional regulation of myriad Mybs in T. vaginalis.
Figure 2. Stereoview of detailed binding interactions of FHA1-pT(Mdt1) (a) in comparisons with th... more Figure 2. Stereoview of detailed binding interactions of FHA1-pT(Mdt1) (a) in comparisons with those of FHA1-pT(Rad9) (b). The ionic interactions involving phosphate group, hydrophobic interactions, and comparable electrostatic/amino-aromatic interactions involving R83 are indicated in red, black, and green, respectively.
The lipoyl-bearing domain (LBD) of the transacylase (E2) subunit of the branched-chain ␣-keto aci... more The lipoyl-bearing domain (LBD) of the transacylase (E2) subunit of the branched-chain ␣-keto acid dehydrogenase complex plays a central role in substrate channeling in this mitochondrial multienzyme complex. We have employed multidimensional heteronuclear NMR techniques to determine the structure and dynamics of the LBD of the human branched-chain ␣-keto acid dehydrogenase complex (hbLBD). Similar to LBD from other members of the ␣-keto acid dehydrogenase family, the solution structure of hbLBD is a flattened -barrel formed by two four-stranded antiparallel -sheets. The lipoyl Lys 44 residue resides at the tip of a -hairpin comprising a sharp type I -turn and the two connecting -strands 4 and 5. A prominent V-shaped groove formed by a surface loop, L1, connecting 1and 2-strands and the lipoyl lysine -hairpin constitutes the functional pocket. We further applied reduced spectral density functions formalism to extract dynamic information of hbLBD from 15 N-T 1 , 15 N-T 2 , and (1 H-15 N) nuclear Overhauser effect data obtained at 600 MHz. The results showed that residues surrounding the lipoyl lysine region comprising the L1 loop and the Lys 44 -turn are highly flexible, whereas -sheet S1 appears to display a slow conformational exchange process.
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Papers by Chi-Fon Chang