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BBA - Proteins and Proteomics (v.1824, #7)
Involvement of Ser94 in RNase HIII from Chlamydophila pneumoniae in the recognition of a single ribonucleotide misincorporated into double-stranded DNA by Zheng Lu; Jingli Hou; You Wang; Jianhua Liu (pp. 859-865).
We recently provided the first report that RNase HIII can cleave a DNA-rN1-DNA/DNA substrate (rN1, one ribonucleotide) in vitro. In the present study, mutagenesis analyses and molecular dynamics (MD) simulations were performed on RNase HIII from Chlamydophila pneumoniae AR39 (CpRNase HIII). Our results elucidate the mechanism of ribonucleotide recognition employed by CpRNase HIII, indicating that the G95/K96/G97 motif of CpRNase HIII represents the main surface interacting with single ribonucleotides, in a manner similar to that of the GR(K)G motif of RNase HIIs. However, CpRNase HIII lacks the specific tyrosine required for RNase HII to recognize single ribonucleotides in double-stranded DNA (dsDNA). Interestingly, MD shows that Ser94 of CpRNase HIII forms a stable hydrogen bond with the deoxyribonucleotide at the (5')RNA–DNA(3') junction, moving this nucleotide away from the chimeric ribonucleotide. This movement appears to deform the nucleic acid backbone at the RNA–DNA junction and allows the ribonucleotide to interact with the GKG motif. Based on the inferences drawn from MD simulations, biochemical results indicated that Ser94 was necessary for catalytic activity on the DNA-rN1-DNA/DNA substrate; mutant S94V could bind this substrate but exhibited no cleavage. Mismatches opposite the single ribonucleotide misincorporated in dsDNA inhibited cleavage by CpRNase HIII to varying degrees but did not interfere with CpRNase/substrate binding. Further MD results implied that mismatches impair the interaction between Ser94 and the deoxyribonucleotide at the RNA–DNA junction. Consequently, recognition of the misincorporated ribonucleotide was disturbed. Our results may help elucidate the distinct substrate-recognition properties of different RNase Hs.► We construct a series of mutants of CpRNase HIII. ► We model the structure of CpRNase HIII complexed with DNA-rN1-DNA/DNA substrate. ► We elucidate the mechanism of ribonucleotide recognition employed by CpRNase HIII. ► Ser94 of CpRNase HIII plays an important role in recognizing single ribonucleotide.
Keywords: Abbreviations; RNase H; ribonuclease H; C. pneumoniae; Chlamydophila pneumoniae; CpRNase; Chlamydophila pneumoniae; RNase; FAM; carboxyfluorescein; E. coli; Escherichia coli; Cntl; control; dsDNA; double-stranded DNA; MD; molecular dynamics Chlamydophila pneumoniae; ribonuclease HIII; serine; molecular dynamics simulation
Involvement of Ser94 in RNase HIII from Chlamydophila pneumoniae in the recognition of a single ribonucleotide misincorporated into double-stranded DNA by Zheng Lu; Jingli Hou; You Wang; Jianhua Liu (pp. 859-865).
We recently provided the first report that RNase HIII can cleave a DNA-rN1-DNA/DNA substrate (rN1, one ribonucleotide) in vitro. In the present study, mutagenesis analyses and molecular dynamics (MD) simulations were performed on RNase HIII from Chlamydophila pneumoniae AR39 (CpRNase HIII). Our results elucidate the mechanism of ribonucleotide recognition employed by CpRNase HIII, indicating that the G95/K96/G97 motif of CpRNase HIII represents the main surface interacting with single ribonucleotides, in a manner similar to that of the GR(K)G motif of RNase HIIs. However, CpRNase HIII lacks the specific tyrosine required for RNase HII to recognize single ribonucleotides in double-stranded DNA (dsDNA). Interestingly, MD shows that Ser94 of CpRNase HIII forms a stable hydrogen bond with the deoxyribonucleotide at the (5')RNA–DNA(3') junction, moving this nucleotide away from the chimeric ribonucleotide. This movement appears to deform the nucleic acid backbone at the RNA–DNA junction and allows the ribonucleotide to interact with the GKG motif. Based on the inferences drawn from MD simulations, biochemical results indicated that Ser94 was necessary for catalytic activity on the DNA-rN1-DNA/DNA substrate; mutant S94V could bind this substrate but exhibited no cleavage. Mismatches opposite the single ribonucleotide misincorporated in dsDNA inhibited cleavage by CpRNase HIII to varying degrees but did not interfere with CpRNase/substrate binding. Further MD results implied that mismatches impair the interaction between Ser94 and the deoxyribonucleotide at the RNA–DNA junction. Consequently, recognition of the misincorporated ribonucleotide was disturbed. Our results may help elucidate the distinct substrate-recognition properties of different RNase Hs.► We construct a series of mutants of CpRNase HIII. ► We model the structure of CpRNase HIII complexed with DNA-rN1-DNA/DNA substrate. ► We elucidate the mechanism of ribonucleotide recognition employed by CpRNase HIII. ► Ser94 of CpRNase HIII plays an important role in recognizing single ribonucleotide.
Keywords: Abbreviations; RNase H; ribonuclease H; C. pneumoniae; Chlamydophila pneumoniae; CpRNase; Chlamydophila pneumoniae; RNase; FAM; carboxyfluorescein; E. coli; Escherichia coli; Cntl; control; dsDNA; double-stranded DNA; MD; molecular dynamics Chlamydophila pneumoniae; ribonuclease HIII; serine; molecular dynamics simulation
Structural dynamics of proximal heme pocket in HemAT- Bs associated with oxygen dissociation by Yuu Yoshida; Haruto Ishikawa; Shigetoshi Aono; Yasuhisa Mizutani (pp. 866-872).
HemAT from Bacillus subtilis (HemAT- Bs) is a heme-containing O2 sensor protein that acts as a chemotactic signal transducer. Binding of O2 to the heme in the sensor domain of HemAT- Bs induces a conformational change in the protein matrix, and this is transmitted to a signaling domain. To characterize the specific mechanism of O2-dependent conformational changes in HemAT- Bs, we investigated time-resolved resonance Raman spectra of the truncated sensor domain and the full-length HemAT- Bs upon O2 and CO dissociation. A comparison between the O2 and CO complexes provides insights on O2/CO discrimination in HemAT- Bs. While no spectral changes upon CO dissociation were observed in our experimental time window between 10ns and 100μs, the band position of the stretching mode between the heme iron and the proximal histidine, ν(Fe–His), for the O2-dissociated HemAT- Bs was lower than that for the deoxy form on time-resolved resonance Raman spectra. This spectral change specific to O2 dissociation would be associated with the O2/CO discrimination in HemAT- Bs. We also compared the results obtained for the truncated sensor domain and the full-length HemAT- Bs, which showed that the structural dynamics related to O2 dissociation for the full-length HemAT- Bs are faster than those for the sensor domain HemAT- Bs. This indicates that the heme proximal structural dynamics upon O2 dissociation are coupled with signal transduction in HemAT- Bs.Display Omitted► Ligand-dependent changes were observed in time-resolved resonance Raman spectra. ► The signaling domain was found to affect the structural change in the heme pocket. ► A coupling mechanism between the heme pocket and the signaling domain was proposed.
Keywords: HemAT; Resonance Raman spectroscopy; Heme oxygen sensor; Signal transduction
Structural dynamics of proximal heme pocket in HemAT- Bs associated with oxygen dissociation by Yuu Yoshida; Haruto Ishikawa; Shigetoshi Aono; Yasuhisa Mizutani (pp. 866-872).
HemAT from Bacillus subtilis (HemAT- Bs) is a heme-containing O2 sensor protein that acts as a chemotactic signal transducer. Binding of O2 to the heme in the sensor domain of HemAT- Bs induces a conformational change in the protein matrix, and this is transmitted to a signaling domain. To characterize the specific mechanism of O2-dependent conformational changes in HemAT- Bs, we investigated time-resolved resonance Raman spectra of the truncated sensor domain and the full-length HemAT- Bs upon O2 and CO dissociation. A comparison between the O2 and CO complexes provides insights on O2/CO discrimination in HemAT- Bs. While no spectral changes upon CO dissociation were observed in our experimental time window between 10ns and 100μs, the band position of the stretching mode between the heme iron and the proximal histidine, ν(Fe–His), for the O2-dissociated HemAT- Bs was lower than that for the deoxy form on time-resolved resonance Raman spectra. This spectral change specific to O2 dissociation would be associated with the O2/CO discrimination in HemAT- Bs. We also compared the results obtained for the truncated sensor domain and the full-length HemAT- Bs, which showed that the structural dynamics related to O2 dissociation for the full-length HemAT- Bs are faster than those for the sensor domain HemAT- Bs. This indicates that the heme proximal structural dynamics upon O2 dissociation are coupled with signal transduction in HemAT- Bs.Display Omitted► Ligand-dependent changes were observed in time-resolved resonance Raman spectra. ► The signaling domain was found to affect the structural change in the heme pocket. ► A coupling mechanism between the heme pocket and the signaling domain was proposed.
Keywords: HemAT; Resonance Raman spectroscopy; Heme oxygen sensor; Signal transduction
FTIR and XPS studies of protein adsorption onto functionalized bioactive glass by C. Gruian; E. Vanea; S. Simon; V. Simon (pp. 873-881).
Adsorption and structural changes that occur upon interaction between methemoglobin (MetHb) and 5-methyl-aminomethyl-uridine forming enzyme (MnmE) with the surface of a bioactive glass (BG) were investigated by Fourier Transform Infrared (FTIR) spectroscopy and X-ray Photoelectron Spectroscopy (XPS). The effect of glutaraldehyde (GA) as a coupling agent for protein adsorption on BG was also investigated. The comparative analysis of FTIR spectra recorded from lyophilized proteins and from bioactive glass surface after protein adsorption was considered in order to obtain information about the changes in the secondary structure of the proteins. XPS data were used to determine the surface coverage. The unfolding of adsorbed proteins due to interactions between the internal hydrophobic protein domains and the hydrophobic BG surface was evidenced. After adsorption, the amount of α-helix decreases and less ordered structures (turns, random coils and aggregates) are preponderant. These changes are less pronounced on the BG functionalized with GA, suggesting that the treatment with GA preserves significantly larger amounts of α-helices in the structure of both proteins after adsorption.► Methemoglobin and MnmE attachment on bioactive glasses (BG) ► Enhancement of protein adherence by BG functionalization with APTS and GA ► Unfolding of proteins adsorbed onto BG particles ► Unfolding is less pronounced on the BG functionalized with GA.
Keywords: Protein adsorption; MetHb; MnmE; Bioactive glass; FTIR; XPS
FTIR and XPS studies of protein adsorption onto functionalized bioactive glass by C. Gruian; E. Vanea; S. Simon; V. Simon (pp. 873-881).
Adsorption and structural changes that occur upon interaction between methemoglobin (MetHb) and 5-methyl-aminomethyl-uridine forming enzyme (MnmE) with the surface of a bioactive glass (BG) were investigated by Fourier Transform Infrared (FTIR) spectroscopy and X-ray Photoelectron Spectroscopy (XPS). The effect of glutaraldehyde (GA) as a coupling agent for protein adsorption on BG was also investigated. The comparative analysis of FTIR spectra recorded from lyophilized proteins and from bioactive glass surface after protein adsorption was considered in order to obtain information about the changes in the secondary structure of the proteins. XPS data were used to determine the surface coverage. The unfolding of adsorbed proteins due to interactions between the internal hydrophobic protein domains and the hydrophobic BG surface was evidenced. After adsorption, the amount of α-helix decreases and less ordered structures (turns, random coils and aggregates) are preponderant. These changes are less pronounced on the BG functionalized with GA, suggesting that the treatment with GA preserves significantly larger amounts of α-helices in the structure of both proteins after adsorption.► Methemoglobin and MnmE attachment on bioactive glasses (BG) ► Enhancement of protein adherence by BG functionalization with APTS and GA ► Unfolding of proteins adsorbed onto BG particles ► Unfolding is less pronounced on the BG functionalized with GA.
Keywords: Protein adsorption; MetHb; MnmE; Bioactive glass; FTIR; XPS
Role of remote scaffolding residues in the inhibitory loop pre-organization, flexibility, rigidification and enzyme inhibition of serine protease inhibitors by Sudip Majumder; Susmita Khamrui; Jhimli Dasgupta; Jiban K. Dattagupta; Udayaditya Sen (pp. 882-890).
Canonical serine protease inhibitors interact with cognate enzymes through the P3–P2′ region of the inhibitory loop while its scaffold hardly makes any contact. Neighboring scaffolding residues like Arginines or Asparagine shape-up the inhibitory loop and favor the resynthesis of cleaved scissile bond. However, role of remote scaffolding residues, which are not involved in religation, was not properly explored. Crystal structures of two engineered winged bean chymotrypsin inhibitor (WCI) complexed with Bovine trypsin (BPT) namely L65R-WCI:BPT and F64Y/L65R-WCI:BPT show that the inhibitory loop of these engineered inhibitors are recognized and rigidified properly at the enzyme active site like other strong trypsin inhibitors. Chimeric protein ETIL-WCIS, having a loop of Erythrina caffra Trypsin Inhibitor, ETI on the scaffold of WCI, was previously shown to behave like substrate. Non-canonical structure of the inhibitory loop and its flexibility are attributed to the presence of smaller scaffolding residues which cannot act as barrier to the inhibitory loop like in ETI. Double mutant A76R/L115Y-(ETIL-WCIS), where the barrier is reintroduced on ETIL-WCIS, shows regaining of inhibitory activity. The structure of A76R/L115Y-(ETIL-WCIS) along with L65R-WCI:BPT and F64Y/L65R-WCI:BPT demonstrate here that the lost canonical conformation of the inhibitory loop is fully restored and loop flexibility is dramatically reduced. Therefore, residues at the inhibitory loop interact with the enzyme playing the primary role in recognition and binding but scaffolding residues having no direct interaction with the enzyme are crucial for rigidification event and the inhibitory potency. B-factor analysis indicates that the amount of inhibitory loop rigidification varies between different inhibitor families.► Rigidification of the inhibitory loop is a primary requisite for proper inhibition. ► Inhibitory loops get stabilized to form complexes with their cognate proteases. ► Remote scaffolding residues play a crucial role in the process of rigidification.
Keywords: Abbreviations; WCI; Winged bean chymotrypsin inhibitor; ECI; Erythrina variegate; chymotrypsin inhibitor; ETI; Erythrina caffra; trypsin inhibitor; STI; soybean trypsin inhibitor; BPTI; bovine pancreatic trypsin inhibitor; ETI; L; -WCI; S; chimera having loop of ETI on the scaffold of WCI; ETI; L; -WCI; S; chimera having loop of STI on the scaffold of WCI; BPT; bovine pancreatic trypsin; PPT; porcine pancreatic trypsin; r.m.s.d.; root mean square deviation; PDB; Protein Data Bank; SDM; site directed mutagenesisChimeric trypsin inhibitor; Loop and scaffold; Crystallography; Inhibitor loop flexibility; Loop rigidification
Role of remote scaffolding residues in the inhibitory loop pre-organization, flexibility, rigidification and enzyme inhibition of serine protease inhibitors by Sudip Majumder; Susmita Khamrui; Jhimli Dasgupta; Jiban K. Dattagupta; Udayaditya Sen (pp. 882-890).
Canonical serine protease inhibitors interact with cognate enzymes through the P3–P2′ region of the inhibitory loop while its scaffold hardly makes any contact. Neighboring scaffolding residues like Arginines or Asparagine shape-up the inhibitory loop and favor the resynthesis of cleaved scissile bond. However, role of remote scaffolding residues, which are not involved in religation, was not properly explored. Crystal structures of two engineered winged bean chymotrypsin inhibitor (WCI) complexed with Bovine trypsin (BPT) namely L65R-WCI:BPT and F64Y/L65R-WCI:BPT show that the inhibitory loop of these engineered inhibitors are recognized and rigidified properly at the enzyme active site like other strong trypsin inhibitors. Chimeric protein ETIL-WCIS, having a loop of Erythrina caffra Trypsin Inhibitor, ETI on the scaffold of WCI, was previously shown to behave like substrate. Non-canonical structure of the inhibitory loop and its flexibility are attributed to the presence of smaller scaffolding residues which cannot act as barrier to the inhibitory loop like in ETI. Double mutant A76R/L115Y-(ETIL-WCIS), where the barrier is reintroduced on ETIL-WCIS, shows regaining of inhibitory activity. The structure of A76R/L115Y-(ETIL-WCIS) along with L65R-WCI:BPT and F64Y/L65R-WCI:BPT demonstrate here that the lost canonical conformation of the inhibitory loop is fully restored and loop flexibility is dramatically reduced. Therefore, residues at the inhibitory loop interact with the enzyme playing the primary role in recognition and binding but scaffolding residues having no direct interaction with the enzyme are crucial for rigidification event and the inhibitory potency. B-factor analysis indicates that the amount of inhibitory loop rigidification varies between different inhibitor families.► Rigidification of the inhibitory loop is a primary requisite for proper inhibition. ► Inhibitory loops get stabilized to form complexes with their cognate proteases. ► Remote scaffolding residues play a crucial role in the process of rigidification.
Keywords: Abbreviations; WCI; Winged bean chymotrypsin inhibitor; ECI; Erythrina variegate; chymotrypsin inhibitor; ETI; Erythrina caffra; trypsin inhibitor; STI; soybean trypsin inhibitor; BPTI; bovine pancreatic trypsin inhibitor; ETI; L; -WCI; S; chimera having loop of ETI on the scaffold of WCI; ETI; L; -WCI; S; chimera having loop of STI on the scaffold of WCI; BPT; bovine pancreatic trypsin; PPT; porcine pancreatic trypsin; r.m.s.d.; root mean square deviation; PDB; Protein Data Bank; SDM; site directed mutagenesisChimeric trypsin inhibitor; Loop and scaffold; Crystallography; Inhibitor loop flexibility; Loop rigidification
Effects of Group 3 LEA protein model peptides on desiccation-induced protein aggregation by Takao Furuki; Tempei Shimizu; Sohini Chakrabortee; Kentarou Yamakawa; Rie Hatanaka; Tsuyoshi Takahashi; Takahiro Kikawada; Takashi Okuda; Hisakazu Mihara; Alan Tunnacliffe; Minoru Sakurai (pp. 891-897).
Group 3 late embryogenesis abundant (G3LEA) proteins have amino acid sequences with characteristic 11-mer motifs and are known to reduce aggregation of proteins during dehydration. Previously, we clarified the structural and thermodynamic properties of the 11-mer repeating units in G3LEA proteins using synthetic peptides composed of two or four tandem repeats originating from an insect ( Polypedilum vanderplanki), nematodes and plants. The purpose of the present study is to test the utility of such 22-mer peptides as protective reagents for aggregation-prone proteins. For lysozyme, desiccation-induced aggregation was abrogated by low molar ratios of a 22-mer peptide, PvLEA-22, derived from a P. vanderplanki G3LEA protein sequence. However, an unexpected behavior was noted for the milk protein, α-casein. On drying, the resultant aggregation was significantly suppressed in the presence of PvLEA-22 with its molar ratios>25 relative to α-casein. However, when the molar ratio was <10, aggregation occurred on addition of PvLEA-22 to aqueous solutions of α-casein. Other peptides derived from nematode, plant and randomized G3LEA protein sequences gave similar results. Such an anomalous solubility change in α-casein was shown to be due to a pH shift to ca. 4, a value nearly equal to the isoelectric point (pI) of α-casein, when any of the 22-mer peptides was mixed. These results demonstrate that synthetic peptides derived from G3LEA protein sequences can reduce protein aggregation caused both by desiccation and, at high molar ratios, also by pH effects, and therefore have potential as stabilization reagents.► We synthesized the 22-mer peptides derived from Group 3 LEA protein sequences. ► The 22-mer peptides suppressed desiccation-induced aggregation of lysozyme. ► For dried α-casein, also, the 22-mer peptides had anti-aggregation activities. ► These findings would arise from molecular shielding effects by the 22-mer peptides. ► For α-casein, the 22-mer peptides could also overcome aggregation due to pH effects.
Keywords: Abbreviations; G3LEA; Group 3 late embryogenesis abundant; PvLEA-22, NeLEA-22, PlLEA-22; 22-mer peptides constructed as the model peptides of the LEA proteins originating from insects (; P. vanderplanki; ), nematodes and plants, respectively; A; 340; Absorbance at 340; nmGroup 3 LEA protein; 22-mer model peptide; Molecular shield; Anti-aggregation; Desiccation; pH
Effects of Group 3 LEA protein model peptides on desiccation-induced protein aggregation by Takao Furuki; Tempei Shimizu; Sohini Chakrabortee; Kentarou Yamakawa; Rie Hatanaka; Tsuyoshi Takahashi; Takahiro Kikawada; Takashi Okuda; Hisakazu Mihara; Alan Tunnacliffe; Minoru Sakurai (pp. 891-897).
Group 3 late embryogenesis abundant (G3LEA) proteins have amino acid sequences with characteristic 11-mer motifs and are known to reduce aggregation of proteins during dehydration. Previously, we clarified the structural and thermodynamic properties of the 11-mer repeating units in G3LEA proteins using synthetic peptides composed of two or four tandem repeats originating from an insect ( Polypedilum vanderplanki), nematodes and plants. The purpose of the present study is to test the utility of such 22-mer peptides as protective reagents for aggregation-prone proteins. For lysozyme, desiccation-induced aggregation was abrogated by low molar ratios of a 22-mer peptide, PvLEA-22, derived from a P. vanderplanki G3LEA protein sequence. However, an unexpected behavior was noted for the milk protein, α-casein. On drying, the resultant aggregation was significantly suppressed in the presence of PvLEA-22 with its molar ratios>25 relative to α-casein. However, when the molar ratio was <10, aggregation occurred on addition of PvLEA-22 to aqueous solutions of α-casein. Other peptides derived from nematode, plant and randomized G3LEA protein sequences gave similar results. Such an anomalous solubility change in α-casein was shown to be due to a pH shift to ca. 4, a value nearly equal to the isoelectric point (pI) of α-casein, when any of the 22-mer peptides was mixed. These results demonstrate that synthetic peptides derived from G3LEA protein sequences can reduce protein aggregation caused both by desiccation and, at high molar ratios, also by pH effects, and therefore have potential as stabilization reagents.► We synthesized the 22-mer peptides derived from Group 3 LEA protein sequences. ► The 22-mer peptides suppressed desiccation-induced aggregation of lysozyme. ► For dried α-casein, also, the 22-mer peptides had anti-aggregation activities. ► These findings would arise from molecular shielding effects by the 22-mer peptides. ► For α-casein, the 22-mer peptides could also overcome aggregation due to pH effects.
Keywords: Abbreviations; G3LEA; Group 3 late embryogenesis abundant; PvLEA-22, NeLEA-22, PlLEA-22; 22-mer peptides constructed as the model peptides of the LEA proteins originating from insects (; P. vanderplanki; ), nematodes and plants, respectively; A; 340; Absorbance at 340; nmGroup 3 LEA protein; 22-mer model peptide; Molecular shield; Anti-aggregation; Desiccation; pH
Deletional studies to investigate the functional role of a dynamic loop region of alkanesulfonate monooxygenase by Jingyuan Xiong; Holly R. Ellis (pp. 898-906).
Several bacterial organisms rely on the two-component alkanesulfonate monooxygenase system for the acquisition of organosulfonate compounds when inorganic sulfur is limiting in the environment. This system is comprised of an FMN reductase (SsuE) that supplies reduced flavin to the alkanesulfonate monooxygenase (SsuD). Desulfonation of alkanesulfonates by SsuD is catalyzed through the activation of dioxygen by reduced flavin. The three-dimensional structure of SsuD exists as a TIM-barrel fold with several discrete insertion regions. An extensive insertion region near the putative active site was disordered in the SsuD structure, suggesting the importance of protein dynamics in the desulfonation mechanism. Three variants containing a partial deletion of the loop region were constructed to evaluate the functional properties of this region. There were no overall gross changes in secondary structure for the three SsuD deletion variants compared to wild-type SsuD, but each variant was found to be catalytically inactive. The deletion variants were unable to undergo the conformational changes necessary for catalysis even though they were able to bind reduced flavin. Rapid kinetic analyses monitoring the reductive and oxidative half-reactions indicated that the SsuD deletion variants failed to protect reduced flavin from unproductive oxidation. These studies define the importance of dynamic loop region for protection and stabilization of reduced flavin and reaction intermediates.► SsuD variants with a shortened unstructured loop region are structurally intact. ► These SsuD variants are able to bind FMNH2 substrate, but are inactive. ► These SsuD variants cannot undergo conformational changes necessary for catalysis. ► The unstructured loop protects FMNH2 and stabilizes reaction intermediates. ► The SsuD unstructured loop may participate in reduced flavin transfer.
Keywords: Abbreviations; DTNB; 5,5-dithiobis(2-nitrobenzoic acid); FMN; flavin mononucleotide; FMNH; 2; reduced flavin mononucleotide; NAD(P)H; nicotinamide adenine dinucleotide (phosphate); (NTA); Ni-nitrilotriacetate; (PMSF); phenylmethylsulfonyl fluoride; SsuE; alkanesulfonate flavin reductase; SsuD; alkanesulfonate monooxygenase; TIM; triosephosphate isomeraseFMN; SsuD; Alkanesulfonate monooxygenase; TIM-barrel fold; Mobile loop; Sulfur
Deletional studies to investigate the functional role of a dynamic loop region of alkanesulfonate monooxygenase by Jingyuan Xiong; Holly R. Ellis (pp. 898-906).
Several bacterial organisms rely on the two-component alkanesulfonate monooxygenase system for the acquisition of organosulfonate compounds when inorganic sulfur is limiting in the environment. This system is comprised of an FMN reductase (SsuE) that supplies reduced flavin to the alkanesulfonate monooxygenase (SsuD). Desulfonation of alkanesulfonates by SsuD is catalyzed through the activation of dioxygen by reduced flavin. The three-dimensional structure of SsuD exists as a TIM-barrel fold with several discrete insertion regions. An extensive insertion region near the putative active site was disordered in the SsuD structure, suggesting the importance of protein dynamics in the desulfonation mechanism. Three variants containing a partial deletion of the loop region were constructed to evaluate the functional properties of this region. There were no overall gross changes in secondary structure for the three SsuD deletion variants compared to wild-type SsuD, but each variant was found to be catalytically inactive. The deletion variants were unable to undergo the conformational changes necessary for catalysis even though they were able to bind reduced flavin. Rapid kinetic analyses monitoring the reductive and oxidative half-reactions indicated that the SsuD deletion variants failed to protect reduced flavin from unproductive oxidation. These studies define the importance of dynamic loop region for protection and stabilization of reduced flavin and reaction intermediates.► SsuD variants with a shortened unstructured loop region are structurally intact. ► These SsuD variants are able to bind FMNH2 substrate, but are inactive. ► These SsuD variants cannot undergo conformational changes necessary for catalysis. ► The unstructured loop protects FMNH2 and stabilizes reaction intermediates. ► The SsuD unstructured loop may participate in reduced flavin transfer.
Keywords: Abbreviations; DTNB; 5,5-dithiobis(2-nitrobenzoic acid); FMN; flavin mononucleotide; FMNH; 2; reduced flavin mononucleotide; NAD(P)H; nicotinamide adenine dinucleotide (phosphate); (NTA); Ni-nitrilotriacetate; (PMSF); phenylmethylsulfonyl fluoride; SsuE; alkanesulfonate flavin reductase; SsuD; alkanesulfonate monooxygenase; TIM; triosephosphate isomeraseFMN; SsuD; Alkanesulfonate monooxygenase; TIM-barrel fold; Mobile loop; Sulfur
A novel serine protease with human fibrino(geno)lytic activities from Artocarpus heterophyllus latex by Jaruwan Siritapetawee; Kanjana Thumanu; Punchapat Sojikul; Sompong Thammasirirak (pp. 907-912).
A protease was isolated and purified from Artocarpus heterophyllus (jackfruit) latex and designated as a 48-kDa antimicrobial protease (AMP48) in a previous publication. In this work, the enzyme was characterized for more biochemical and medicinal properties. Enzyme activity of AMP48 was strongly inhibited by phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, indicating that the enzyme was a plant serine protease. The N-terminal amino acid sequences (A-Q-E-G-G-K-D-D-D-G-G) of AMP48 had no sequence similarity matches with any sequence databases of BLAST search and other plant serine protease. The secondary structure of this enzyme was composed of high α-helix (51%) and low β-sheet (9%). AMP48 had fibrinogenolytic activity with maximal activity between 55 and 60°C at pH 8. The enzyme efficiently hydrolyzed α followed by partially hydrolyzed β and γ subunits of human fibrinogen. In addition, the fibrinolytic activity was observed through the degradation products by SDS-PAGE and emphasized its activity by monitoring the alteration of secondary structure of fibrin clot after enzyme digestion using ATR-FTIR spectroscopy. This study presented the potential role to use AMP48 as antithrombotic for treatment thromboembolic disorders such as strokes, pulmonary emboli and deep vein thrombosis.Display Omitted► A novel protease (AMP48) was purified from Artocarpus heterophyllus latex. ► The enzyme efficiently hydrolyzed a subunit of human fibrinogen. ► Fibrinolytic activity of AMP48 was emphasized using ATR-FTIR spectroscopy. ► This work presented a potential to use AMP48 for treatment thromboembolic disorders.
Keywords: Artocarpus heterophyllus; Jackfruit; Latex; Serine protease
A novel serine protease with human fibrino(geno)lytic activities from Artocarpus heterophyllus latex by Jaruwan Siritapetawee; Kanjana Thumanu; Punchapat Sojikul; Sompong Thammasirirak (pp. 907-912).
A protease was isolated and purified from Artocarpus heterophyllus (jackfruit) latex and designated as a 48-kDa antimicrobial protease (AMP48) in a previous publication. In this work, the enzyme was characterized for more biochemical and medicinal properties. Enzyme activity of AMP48 was strongly inhibited by phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, indicating that the enzyme was a plant serine protease. The N-terminal amino acid sequences (A-Q-E-G-G-K-D-D-D-G-G) of AMP48 had no sequence similarity matches with any sequence databases of BLAST search and other plant serine protease. The secondary structure of this enzyme was composed of high α-helix (51%) and low β-sheet (9%). AMP48 had fibrinogenolytic activity with maximal activity between 55 and 60°C at pH 8. The enzyme efficiently hydrolyzed α followed by partially hydrolyzed β and γ subunits of human fibrinogen. In addition, the fibrinolytic activity was observed through the degradation products by SDS-PAGE and emphasized its activity by monitoring the alteration of secondary structure of fibrin clot after enzyme digestion using ATR-FTIR spectroscopy. This study presented the potential role to use AMP48 as antithrombotic for treatment thromboembolic disorders such as strokes, pulmonary emboli and deep vein thrombosis.Display Omitted► A novel protease (AMP48) was purified from Artocarpus heterophyllus latex. ► The enzyme efficiently hydrolyzed a subunit of human fibrinogen. ► Fibrinolytic activity of AMP48 was emphasized using ATR-FTIR spectroscopy. ► This work presented a potential to use AMP48 for treatment thromboembolic disorders.
Keywords: Artocarpus heterophyllus; Jackfruit; Latex; Serine protease
Constant pH Molecular Dynamics (CpHMD) and mutation studies: Insights into AaegOBP1 pH-induced ligand releasing mechanism by Wen-Ting Chu; Yun-Jian Wu; Ji-Long Zhang; Qing-Chuan Zheng; Lin Chen; Qiao Xue; Hong-Xing Zhang (pp. 913-918).
AaegOBP1, isolated from the male and female antenna of yellow fever mosquitoes, may serve as crucial molecular targets for the development of mosquitoes’ attractants and for the control of mosquito populations. Nowadays crystal structures of AaegOBP1 in the neutral environment have been obtained, whereas little research is focused on the conformational change of AaegOBP1 in the acid solution. In our study, the conformational change and the ligand bound poses in different solution pH were investigated using constant pH molecular dynamics (CpHMD) as well as mutation studies. Results demonstrate that the protein changes dramatically in low pH solution and that the pH-sensing triad (Arg23-Tyr54-Ile125) plays an indispensable role in the structural stability and ligand binding. In addition, we have proved that the residue Arg23 is the most important one of the pH-sensing triad. This work could provide more penetrating understanding of the pH-induced ligand-releasing mechanism.Display Omitted► we perform CpHMD simulations to explain the conformational change from pH 7 to 5. ► pKa prediction of AaegOBP1 is calculated after MD simulation.
Keywords: AaegOBP1; CpHMD; pH dependent; Odorant binding protein; pH-induced ligand releasing
Constant pH Molecular Dynamics (CpHMD) and mutation studies: Insights into AaegOBP1 pH-induced ligand releasing mechanism by Wen-Ting Chu; Yun-Jian Wu; Ji-Long Zhang; Qing-Chuan Zheng; Lin Chen; Qiao Xue; Hong-Xing Zhang (pp. 913-918).
AaegOBP1, isolated from the male and female antenna of yellow fever mosquitoes, may serve as crucial molecular targets for the development of mosquitoes’ attractants and for the control of mosquito populations. Nowadays crystal structures of AaegOBP1 in the neutral environment have been obtained, whereas little research is focused on the conformational change of AaegOBP1 in the acid solution. In our study, the conformational change and the ligand bound poses in different solution pH were investigated using constant pH molecular dynamics (CpHMD) as well as mutation studies. Results demonstrate that the protein changes dramatically in low pH solution and that the pH-sensing triad (Arg23-Tyr54-Ile125) plays an indispensable role in the structural stability and ligand binding. In addition, we have proved that the residue Arg23 is the most important one of the pH-sensing triad. This work could provide more penetrating understanding of the pH-induced ligand-releasing mechanism.Display Omitted► we perform CpHMD simulations to explain the conformational change from pH 7 to 5. ► pKa prediction of AaegOBP1 is calculated after MD simulation.
Keywords: AaegOBP1; CpHMD; pH dependent; Odorant binding protein; pH-induced ligand releasing
Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K by Ryuichiro Suzuki; Kazue Terasawa; Keitarou Kimura; Zui Fujimoto; Mitsuru Momma; Mikihiko Kobayashi; Atsuo Kimura; Kazumi Funane (pp. 919-924).
Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤50°C) was 10°C higher than that for CITase-T3040 (≤40°C); the kcat/ KM value of CITase-598K was approximately two times higher (32.2s−1mM−1) than that of CITase-T3040 (17.8s−1mM−1). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.Display Omitted► CITse-598K is a novel cycloisomaltooligosaccharide glucanotransferase. ► CITase-598K exhibited higher activity and thermal stability. ► The major product of CITase-598K was cycloisomaltoheptaose. ► The minimum size of substrate for CITase is isomaltotetraose. ► Asp144, Asp269, and Glu341 of CITase-598K are catalytically important.
Keywords: Abbreviations; CBM; carbohydrate-binding module; CI; cycloisomaltooligosaccharide; CITase; CI glucanotransferase; CITase-598K; CITase from; Paenibacillus; sp. 598K; CITase-T3040; CITase from; Bacillus circulans; T-3040; DP; degree of polymerization; EDTA; ethylenediaminetetraacetic acid; EGTA; ethylene glycol tetraacetic acid; GH; glycoside hydrolase; HPLC; high-performance liquid chromatography; IG; isomaltooligosaccharide; IG2; isomaltose; IG3; isomaltotriose; IG4; isomaltotetraose; IG5; isomaltopentaose; IG6; isomaltohexaose; IG7; isomaltoheptaose; PCR; polymerase chain reaction; PsDex; dextranase from; Paenibacillus; sp; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SmuDXA; dextranase from; Streptococcus mutansCycloisomaltooligosaccharide; Cyclodextran; Cycloisomaltooligosaccharide glucanotransferase; Catalytic residue
Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K by Ryuichiro Suzuki; Kazue Terasawa; Keitarou Kimura; Zui Fujimoto; Mitsuru Momma; Mikihiko Kobayashi; Atsuo Kimura; Kazumi Funane (pp. 919-924).
Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤50°C) was 10°C higher than that for CITase-T3040 (≤40°C); the kcat/ KM value of CITase-598K was approximately two times higher (32.2s−1mM−1) than that of CITase-T3040 (17.8s−1mM−1). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.Display Omitted► CITse-598K is a novel cycloisomaltooligosaccharide glucanotransferase. ► CITase-598K exhibited higher activity and thermal stability. ► The major product of CITase-598K was cycloisomaltoheptaose. ► The minimum size of substrate for CITase is isomaltotetraose. ► Asp144, Asp269, and Glu341 of CITase-598K are catalytically important.
Keywords: Abbreviations; CBM; carbohydrate-binding module; CI; cycloisomaltooligosaccharide; CITase; CI glucanotransferase; CITase-598K; CITase from; Paenibacillus; sp. 598K; CITase-T3040; CITase from; Bacillus circulans; T-3040; DP; degree of polymerization; EDTA; ethylenediaminetetraacetic acid; EGTA; ethylene glycol tetraacetic acid; GH; glycoside hydrolase; HPLC; high-performance liquid chromatography; IG; isomaltooligosaccharide; IG2; isomaltose; IG3; isomaltotriose; IG4; isomaltotetraose; IG5; isomaltopentaose; IG6; isomaltohexaose; IG7; isomaltoheptaose; PCR; polymerase chain reaction; PsDex; dextranase from; Paenibacillus; sp; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SmuDXA; dextranase from; Streptococcus mutansCycloisomaltooligosaccharide; Cyclodextran; Cycloisomaltooligosaccharide glucanotransferase; Catalytic residue
Identification of a Wnt-induced protein complex by affinity proteomics using an antibody that recognizes a sub-population of β-catenin by Meredith J. Layton; Maree C. Faux; Nicole L. Church; Bruno Catimel; Nadia J. Kershaw; Eugene A. Kapp; Cameron Nowell; Janine L. Coates; Antony W. Burgess; Richard J. Simpson (pp. 925-937).
β-catenin is a signaling protein with diverse functions in cell adhesion and Wnt signaling. Although β-catenin has been shown to participate in many protein–protein interactions, it is not clear which combinations of β-catenin-interacting proteins form discrete complexes. We have generated a novel antibody, termed 4B3, which recognizes only a small subset of total cellular β-catenin. Affinity proteomics using 4B3, in combination with subcellular fractionation, has allowed us to define a discrete trimeric complex of β-catenin, α-catenin and the tumor suppressor APC, which forms in the cytoplasm in response to Wnt signaling. Depletion of the limiting component of this complex, APC, implicates the complex in mediating Wnt-induced changes in cell–cell adhesion. APC is also essential for N-terminal phosphorylation of β-catenin within this complex. Each component of β-catenin/APC/α-catenin complex co-exists in other protein complexes, thus use of a selective antibody for affinity proteomics has allowed us to go beyond the generation of a list of potential β-catenin-interacting proteins, and define when and where a specific complex forms.► A novel antibody that binds a subset of total cellular β-catenin was generated. ► Affinity proteomics defines a trimeric complex that forms in response to Wnt3a. ► Subset-specific Abs allow affinity proteomics to go beyond a list of binding proteins
Keywords: Abbreviations; APC; adenomatous polyposis coli; LEF/Tcf; leukemia enhancement factor/T cell factor; CK; casein kinase; GSK; glycogen synthase kinase; L; conditioned media from parental L cells; W; conditioned media from L cells stably transfected with Wnt3a; mAb; monoclonal antibody; HMW; high molecular weight; LMW; low molecular weight; SEC; size-exclusion chromatography; siRNA; short interfering RNA; TX-100; Triton X-100; HBS; HEPES buffered saline; EE; glu or EE epitope tag; DME; Dulbecco's Modified Eagles media; MW; molecular weight; CMC; critical micelle concentration; Ip; immunoprecipitation; pptn; precipitationβ-catenin; Adenomatous polyposis coli; α-catenin; Affinity proteomics; Protein complex; Wnt signaling
Identification of a Wnt-induced protein complex by affinity proteomics using an antibody that recognizes a sub-population of β-catenin by Meredith J. Layton; Maree C. Faux; Nicole L. Church; Bruno Catimel; Nadia J. Kershaw; Eugene A. Kapp; Cameron Nowell; Janine L. Coates; Antony W. Burgess; Richard J. Simpson (pp. 925-937).
β-catenin is a signaling protein with diverse functions in cell adhesion and Wnt signaling. Although β-catenin has been shown to participate in many protein–protein interactions, it is not clear which combinations of β-catenin-interacting proteins form discrete complexes. We have generated a novel antibody, termed 4B3, which recognizes only a small subset of total cellular β-catenin. Affinity proteomics using 4B3, in combination with subcellular fractionation, has allowed us to define a discrete trimeric complex of β-catenin, α-catenin and the tumor suppressor APC, which forms in the cytoplasm in response to Wnt signaling. Depletion of the limiting component of this complex, APC, implicates the complex in mediating Wnt-induced changes in cell–cell adhesion. APC is also essential for N-terminal phosphorylation of β-catenin within this complex. Each component of β-catenin/APC/α-catenin complex co-exists in other protein complexes, thus use of a selective antibody for affinity proteomics has allowed us to go beyond the generation of a list of potential β-catenin-interacting proteins, and define when and where a specific complex forms.► A novel antibody that binds a subset of total cellular β-catenin was generated. ► Affinity proteomics defines a trimeric complex that forms in response to Wnt3a. ► Subset-specific Abs allow affinity proteomics to go beyond a list of binding proteins
Keywords: Abbreviations; APC; adenomatous polyposis coli; LEF/Tcf; leukemia enhancement factor/T cell factor; CK; casein kinase; GSK; glycogen synthase kinase; L; conditioned media from parental L cells; W; conditioned media from L cells stably transfected with Wnt3a; mAb; monoclonal antibody; HMW; high molecular weight; LMW; low molecular weight; SEC; size-exclusion chromatography; siRNA; short interfering RNA; TX-100; Triton X-100; HBS; HEPES buffered saline; EE; glu or EE epitope tag; DME; Dulbecco's Modified Eagles media; MW; molecular weight; CMC; critical micelle concentration; Ip; immunoprecipitation; pptn; precipitationβ-catenin; Adenomatous polyposis coli; α-catenin; Affinity proteomics; Protein complex; Wnt signaling
The mechanism of shared but distinct CSF-1R signaling by the non-homologous cytokines IL-34 and CSF-1 by Heli Liu; Cindy Leo; Xiaoyan Chen; Brian R. Wong; Lewis T. Williams; Haishan Lin; Xiaolin He (pp. 938-945).
Interleukin-34 (IL-34) and colony stimulating factor-1 (CSF-1) both signal through the CSF-1R receptor tyrosine kinase, but they have no sequence homology, and their functions and signaling activities are not identical. We report the crystal structures of mouse IL-34 alone and in complex with the N-terminal three immunoglobulin-like domains (D1-D3) of mouse CSF-1R. IL-34 is structurally related to other helical hematopoietic cytokines, but contains two additional helices integrally associated with the four shared helices. The non-covalently linked IL-34 homodimer recruits two copies of CSF-1R on the sides of the helical bundles, with an overall shape similar to the CSF-1:CSF-1R complex, but the flexible linker between CSF-1R D2 and D3 allows these domains to clamp IL-34 and CSF-1 at different angles. Functional dissection of the IL-34:CSF-1R interface indicates that the hydrophobic interactions, rather than the salt bridge network, dominate the biological activity of IL-34. To degenerately recognize two ligands with completely different surfaces, CSF-1R apparently takes advantage of different subsets of a chemically inert surface that can be tuned to fit different ligand shapes. Differentiated signaling between IL-34 and CSF-1 is likely achieved by the relative thermodynamic independence of IL-34 vs. negative cooperativity of CSF-1 at the receptor-recognition sites, in combination with the difference in hydrophobicity which dictates a more stable IL-34:CSF-1R complex compared to the CSF-1:CSF-1R complex.► Structures of mouse interleukin-34 and its complex with receptor. ► The basis of CSF-1R cross-reactivity towards non-homologous ligands is revealed. ► Functional dissection of the interleukin-34: CSF-1R interface reveals hotspots. ► Conformational flexibility and chemical inertness underline CSF-1R degeneracy.
Keywords: Abbreviations; IL-34; interleukin-34; CSF1; colony stimulating factor-1; CSF-1R; colony stimulating factor-1 receptor; ITC; isothermal titration calorimetryInterleukin-34; Colony stimulating factor-1 receptor; Growth factor; Receptor tyrosine kinase; X-ray crystallography; Ligand/receptor binding
The mechanism of shared but distinct CSF-1R signaling by the non-homologous cytokines IL-34 and CSF-1 by Heli Liu; Cindy Leo; Xiaoyan Chen; Brian R. Wong; Lewis T. Williams; Haishan Lin; Xiaolin He (pp. 938-945).
Interleukin-34 (IL-34) and colony stimulating factor-1 (CSF-1) both signal through the CSF-1R receptor tyrosine kinase, but they have no sequence homology, and their functions and signaling activities are not identical. We report the crystal structures of mouse IL-34 alone and in complex with the N-terminal three immunoglobulin-like domains (D1-D3) of mouse CSF-1R. IL-34 is structurally related to other helical hematopoietic cytokines, but contains two additional helices integrally associated with the four shared helices. The non-covalently linked IL-34 homodimer recruits two copies of CSF-1R on the sides of the helical bundles, with an overall shape similar to the CSF-1:CSF-1R complex, but the flexible linker between CSF-1R D2 and D3 allows these domains to clamp IL-34 and CSF-1 at different angles. Functional dissection of the IL-34:CSF-1R interface indicates that the hydrophobic interactions, rather than the salt bridge network, dominate the biological activity of IL-34. To degenerately recognize two ligands with completely different surfaces, CSF-1R apparently takes advantage of different subsets of a chemically inert surface that can be tuned to fit different ligand shapes. Differentiated signaling between IL-34 and CSF-1 is likely achieved by the relative thermodynamic independence of IL-34 vs. negative cooperativity of CSF-1 at the receptor-recognition sites, in combination with the difference in hydrophobicity which dictates a more stable IL-34:CSF-1R complex compared to the CSF-1:CSF-1R complex.► Structures of mouse interleukin-34 and its complex with receptor. ► The basis of CSF-1R cross-reactivity towards non-homologous ligands is revealed. ► Functional dissection of the interleukin-34: CSF-1R interface reveals hotspots. ► Conformational flexibility and chemical inertness underline CSF-1R degeneracy.
Keywords: Abbreviations; IL-34; interleukin-34; CSF1; colony stimulating factor-1; CSF-1R; colony stimulating factor-1 receptor; ITC; isothermal titration calorimetryInterleukin-34; Colony stimulating factor-1 receptor; Growth factor; Receptor tyrosine kinase; X-ray crystallography; Ligand/receptor binding