Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 21
Filter
Add more filters










Publication year range
1.
Nat Methods ; 20(11): 1729-1738, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37813988

ABSTRACT

Cryo-electron microscopy (cryo-EM) captures snapshots of dynamic macromolecules, collectively illustrating the involved structural landscapes. This provides an exciting opportunity to explore the structural variations of macromolecules under study. However, traditional cryo-EM single-particle analysis often yields static structures. Here we describe OPUS-DSD, an algorithm capable of efficiently reconstructing the structural landscape embedded in cryo-EM data. OPUS-DSD uses a three-dimensional convolutional encoder-decoder architecture trained with cryo-EM images, thereby encoding structural variations into a smooth and easily analyzable low-dimension space. This space can be traversed to reconstruct continuous dynamics or clustered to identify distinct conformations. OPUS-DSD can offer meaningful insights into the structural variations of macromolecules, filling in the gaps left by traditional cryo-EM structural determination, and potentially improves the reconstruction resolution by reliably clustering similar particles within the dataset. These functionalities are especially relevant to the study of highly dynamic biological systems. OPUS-DSD is available at https://github.com/alncat/opusDSD .


Subject(s)
Algorithms , Single Molecule Imaging , Cryoelectron Microscopy/methods , Cluster Analysis , Macromolecular Substances/chemistry
2.
Zhongguo Zhong Yao Za Zhi ; 48(11): 3022-3031, 2023 Jun.
Article in Chinese | MEDLINE | ID: mdl-37381960

ABSTRACT

This study aims to investigate the effect and mechanism of arctigenin(ARC) in the treatment of vascular endothelial injury in rats with pregnancy-induced hypertension(PIH). Fifty SD rats pregnant for 12 days were randomly assigned into a control group, a model group, an ARC group, a rapamycin(RAP, autophagy inducer) group, and an ARC+3-methyladenine(3-MA, autophagy inhibitor) group, with 10 rats in each group. The rats in the other groups except the control group were intraperitoneally injected with nitrosyl-L-arginine methyl ester(50 mg·kg~(-1)·d~(-1)) to establish the PIH model on the 13th day of pregnancy. On the 15th day of pregnancy, the rats in ARC, RAP, and ARC+3-MA groups were intraperitoneally injected with ARC(50 mg·kg~(-1)·d~(-1)), RAP(1 mg·kg~(-1)·d~(-1)), and 3-MA(15 mg·kg~(-1)·d~(-1))+ARC(50 mg·kg~(-1)·d~(-1)), respectively. The pregnant rats in the control group and the model group were intraperitoneally injected with the same amount of normal saline. The blood pressure and 24 h urine protein(24 h-UP) of pregnant rats in each group were measured before and after intervention. Cesarean section was performed to terminate pregnancy on day 21, and the body weight and body length of fetal rats were compared among groups. Hematoxylin-eosin(HE) staining was employed to observe the pathological changes of placenta. The expression of endothelin-1(ET-1) and endothelial nitric oxide synthase(eNOS) in placenta was detected by immunohistochemistry. The serum levels of ET-1 and nitric oxide(NO) were determined with corresponding kits. The expression of microtubule-associated protein 1 light chain 3(LC3), Beclin-1, NOD-like receptor protein 3(NLRP3), apoptosis-associated speck-like protein with CARD domain(ASC), caspase-1, interleukin(IL)-1ß, and IL-18 was determined by immunofluorescence and Western blot. The level of reactive oxygen species(ROS) in placenta was measured by fluorescence staining. The results showed that on day 12 of pregnancy, the blood pressure and 24 h-UP had no significant differences among groups. On days 15, 19, and 21, the blood pressure and 24 h-UP in the model group were higher than those in the control group(P<0.05). On days 19 and 21, the blood pressure and 24 h-UP in ARC group and RAP group were lower than those in the model group(P<0.05), and they were higher in the ARC+3-MA group than in the ARC group(P<0.05). On day 21, the model group had lower body weight and body length of fetal rats(P<0.05), higher serum level of ET-1, and lower serum level of NO(P<0.05) than the control group. Moreover, the placental tissue showed typical pathological damage, down-regulated expression of LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and eNOS(P<0.05), up-regulated expression of ET-1, NLRP3, ASC, caspase-1, IL-1ß, and IL-18(P<0.05), and elevated ROS level. Compared with the model group, ARC and RAP groups showed increased body weight and body length of fetal rats(P<0.05), lowered serum level of ET-1, elevated serum level of NO(P<0.05), reduced pathological damage of placental tissue, up-regulated expression of LC3-Ⅱ/LC3-Ⅰ, Beclin-1, and eNOS(P<0.05), down-regulated expression of ET-1, NLRP3, ASC, caspase-1, IL-1ß, and IL-18(P<0.05), and lowered ROS level. Compared with ARC group, 3-MA reversed the effects of ARC on the above indicators. In conclusion, ARC can inhibit the activation of NLRP3 inflammasome and mitigate vascular endothelial damage in PIH rats by inducing autophagy of vascular endothelial cells.


Subject(s)
Hypertension, Pregnancy-Induced , Female , Pregnancy , Animals , Rats , Humans , Rats, Sprague-Dawley , Hypertension, Pregnancy-Induced/drug therapy , Endothelial Cells , Inflammasomes , Interleukin-18 , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Beclin-1 , Cesarean Section , Reactive Oxygen Species , Placenta , Caspase 1 , Autophagy
3.
PLoS One ; 14(2): e0210749, 2019.
Article in English | MEDLINE | ID: mdl-30730999

ABSTRACT

Globally, human respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in newborns, young children, and the elderly for which there is no vaccine. The RSV fusion (F) glycoprotein is a major target for vaccine development. Here, we describe a novel monoclonal antibody (designated as R4.C6) that recognizes both pre-fusion and post-fusion RSV F, and binds with nanomole affinity to a unique neutralizing site comprised of antigenic sites II and IV on the globular head. A 3.9 Å-resolution structure of RSV F-R4.C6 Fab complex was obtained by single particle cryo-electron microscopy and 3D reconstruction. The structure unraveled detailed interactions of R4.C6 with antigenic site II on one protomer and site IV on a neighboring protomer of post-fusion RSV F protein. These findings significantly further our understanding of the antigenic complexity of the F protein and provide new insights into RSV vaccine design.


Subject(s)
Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , Binding Sites, Antibody , Immunoglobulin Fab Fragments/chemistry , Respiratory Syncytial Viruses/chemistry , Viral Fusion Proteins/chemistry , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Cell Line , Humans , Immunoglobulin Fab Fragments/immunology , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Viruses/immunology , Spodoptera , Viral Fusion Proteins/immunology
4.
Virology ; 513: 98-107, 2018 01 01.
Article in English | MEDLINE | ID: mdl-29055255

ABSTRACT

Influenza pandemic occurs when a new strain from other animal species overcomes the inter-species barriers and supports rapid human-to-human transmission. A critical prerequisite to this process is that hemagglutinin (HA) acquires a few key mutations to switch from avian receptors to human receptors. Previous studies suggest that H1 and H2/H3 HAs use different sets of mutations for the switch. This report shows that HA from the 1918 H1N1 pandemic virus (1918H1 HA) adopts the set of mutations used by H2/H3 HAs in receptor-preference switch when its 130-loop is made similar to those of H2/H3 HAs. Thus, the 130-loop appears to be the key determinant for the different mutations employed by pandemic H1 or H2/H3 HA. The correlation of the mutational routes and the 130-loop as unraveled in this study opens the door for efficient investigation of mutations required by other HA subtypes for inter-human airborne transmission.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Receptors, Virus/metabolism , Crystallography, X-Ray , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Humans , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Binding , Protein Conformation
5.
Proc Natl Acad Sci U S A ; 112(41): 12687-92, 2015 Oct 13.
Article in English | MEDLINE | ID: mdl-26417072

ABSTRACT

Leiomodin (Lmod) is a class of potent tandem-G-actin-binding nucleators in muscle cells. Lmod mutations, deletion, or instability are linked to lethal nemaline myopathy. However, the lack of high-resolution structures of Lmod nucleators in action severely hampered our understanding of their essential cellular functions. Here we report the crystal structure of the actin-Lmod2162-495 nucleus. The structure contains two actin subunits connected by one Lmod2162-495 molecule in a non-filament-like conformation. Complementary functional studies suggest that the binding of Lmod2 stimulates ATP hydrolysis and accelerates actin nucleation and polymerization. The high level of conservation among Lmod proteins in sequence and functions suggests that the mechanistic insights of human Lmod2 uncovered here may aid in a molecular understanding of other Lmod proteins. Furthermore, our structural and mechanistic studies unraveled a previously unrecognized level of regulation in mammalian signal transduction mediated by certain tandem-G-actin-binding nucleators.


Subject(s)
Actin Cytoskeleton/chemistry , Microfilament Proteins/chemistry , Muscle Cells , Muscle Proteins/chemistry , Actin Cytoskeleton/genetics , Animals , Crystallography, X-Ray , Drosophila melanogaster , Humans , Microfilament Proteins/genetics , Muscle Proteins/genetics , Protein Structure, Quaternary , Rabbits , Structure-Activity Relationship
6.
PLoS One ; 10(7): e0134576, 2015.
Article in English | MEDLINE | ID: mdl-26226046

ABSTRACT

In June 2013, the first human infection by avian influenza A(H6N1) virus was reported in Taiwan. This incident raised the concern for possible human epidemics and pandemics from H6 viruses. In this study, we performed structural and functional investigation on the hemagglutinin (HA) proteins of the human-infecting A/Taiwan/2/2013(H6N1) (TW H6) virus and an avian A/chicken/Guangdong/S1311/2010(H6N6) (GD H6) virus that transmitted efficiently in guinea pigs. Our results revealed that in the presence of HA1 Q226, the triad of HA1 S137, E190 and G228 in GD H6 HA allows the binding to both avian- and human-like receptors with a slight preference for avian receptors. Its conservation among the majority of H6 HAs provides an explanation for the broader host range of this subtype. Furthermore, the triad of N137, V190 and S228 in TW H6 HA may alleviate the requirement for a hydrophobic residue at HA1 226 of H2 and H3 HAs when binding to human-like receptors. Consequently, TW H6 HA has a slight preference for human receptors, thus may represent an intermediate towards a complete human adaptation. Importantly, the triad observed in TW H6 HA is detected in 74% H6 viruses isolated from Taiwan in the past 14 years, suggesting an elevated threat of H6 viruses from this region to human health. The novel roles of the triad at HA1 137, 190 and 228 of H6 HA in binding to receptors revealed here may also be used by other HA subtypes to achieve human adaptation, which needs to be further tested in laboratory and closely monitored in field surveillance.


Subject(s)
Hemagglutinins, Viral/genetics , Influenza A virus/ultrastructure , Influenza, Human/virology , Animals , Chickens/virology , Guinea Pigs , Humans , Influenza A virus/isolation & purification , Influenza A virus/physiology , Influenza in Birds/virology , Influenza, Human/epidemiology , Orthomyxoviridae Infections/virology , Phylogeny , Protein Conformation
7.
Virology ; 450-451: 71-83, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24503069

ABSTRACT

Diverged ~4000 years ago, influenza B virus has several important differences from influenza A virus, including lower receptor-binding affinity and highly restricted host range. Based on our prior structural studies, we hypothesized that a single-residue difference in the receptor-binding site of hemagglutinin (HA), Phe-95 in influenza B virus versus Tyr-98 in influenza A/H1-H15, is possibly a key determinant for the low receptor-binding affinity. Here we demonstrate that the mutation Phe95→Tyr in influenza B virus HA restores all three hydrogen bonds made by Tyr-98 in influenza A/H1-15 HA and has the potential to enhance receptor binding. However, the full realization of this potential is influenced by the local environment into which the mutation is introduced. The binding and replication of the recombinant viruses correlate well with the receptor-binding capabilities of HA. These results are discussed in relation to the roles of Phe-95 in receptor binding and pathogenicity of influenza B virus.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Influenza B virus/metabolism , Phenylalanine/metabolism , Receptors, Virus/metabolism , Amino Acid Motifs , Animals , Female , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Humans , Influenza A virus/genetics , Influenza A virus/metabolism , Influenza B virus/chemistry , Influenza B virus/genetics , Influenza B virus/pathogenicity , Mice, Inbred BALB C , Models, Molecular , Phenylalanine/genetics , Protein Binding , Receptors, Virus/genetics , Virulence
8.
Biochemistry ; 53(5): 846-54, 2014 Feb 11.
Article in English | MEDLINE | ID: mdl-24433110

ABSTRACT

Membrane fusion is involved in many fundamental cellular processes and entry of enveloped viruses into host cells. Influenza type A virus HA has long served as a paradigm for mechanistic studies of protein-mediated membrane fusion via large-scale structural rearrangements induced by acidic pH. Here we report the newly determined crystal structure of influenza B virus HA2 in the postfusion state. Together with a large number of previously determined prefusion structures of influenza A and B virus HA and a postfusion structure of influenza A/H3N2 HA2, we identified conserved features that are shared between influenza A and B virus HA in the conformational transition and documented substantial differences that likely influence the detailed mechanisms of this process. Further studies are needed to dissect the effects of these and other structural differences in HA conformational changes and influenza pathogenicity and transmission, which may ultimately expedite the discovery of novel anti-influenza fusion inhibitors.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Influenza B virus/metabolism , Membrane Fusion , Influenza A Virus, H3N2 Subtype/metabolism , Models, Molecular , Protein Conformation
9.
Virology ; 446(1-2): 112-22, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24074573

ABSTRACT

Influenza A and B viruses are responsible for the severe morbidity and mortality worldwide in annual influenza epidemics. Currently circulating influenza B virus belongs to the B/Victoria or B/Yamagata lineage that was diverged from each other about 30-40 years ago. However, a mechanistic understanding of their divergent evolution is still lacking. Here we report the crystal structures of influenza B/Yamanashi/166/1998 hemagglutinin (HA) belonging to B/Yamagata lineage and its complex with the avian-like receptor analogue. Comparison of these structures with those of undiverged and diverged influenza B virus HAs, in conjunction with sequence analysis, reveals the molecular basis for the divergent evolution of influenza B virus HAs. Furthermore, HAs of diverged influenza B virus strains display much stronger molecular interactions with terminal sialic acid of bound receptors, which may allow for a different tissue tropism for current influenza B viruses, for which further investigation is required.


Subject(s)
Evolution, Molecular , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Influenza B virus/chemistry , Crystallography, X-Ray , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Humans , Influenza B virus/genetics , Models, Molecular , Protein Binding , Protein Conformation , Receptors, Virus/metabolism , Sialic Acids/metabolism , Virus Attachment
10.
Cell Rep ; 3(6): 1910-20, 2013 Jun 27.
Article in English | MEDLINE | ID: mdl-23727244

ABSTRACT

Spontaneous nucleation of actin is very inefficient in cells. To overcome this barrier, cells have evolved a set of actin filament nucleators to promote rapid nucleation and polymerization in response to specific stimuli. However, the molecular mechanism of actin nucleation remains poorly understood. This is hindered largely by the fact that actin nucleus, once formed, rapidly polymerizes into filament, thus making it impossible to capture stable multisubunit actin nucleus. Here, we report an effective double-mutant strategy to stabilize actin nucleus by preventing further polymerization. Employing this strategy, we solved the crystal structure of AMPPNP-actin in complex with the first two tandem W domains of Cordon-bleu (Cobl), a potent actin filament nucleator. Further sequence comparison and functional studies suggest that the nucleation mechanism of Cobl is probably shared by the p53 cofactor JMY, but not Spire. Moreover, the double-mutant strategy opens the way for atomic mechanistic study of actin nucleation and polymerization.


Subject(s)
Actin Cytoskeleton/chemistry , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/genetics , Actins/chemistry , Actins/genetics , Actins/metabolism , Amino Acid Sequence , Animals , Crystallography, X-Ray , Drosophila , Humans , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Quaternary , Protein Structure, Tertiary
11.
Proc Natl Acad Sci U S A ; 107(25): 11352-7, 2010 Jun 22.
Article in English | MEDLINE | ID: mdl-20534430

ABSTRACT

Voltage-dependent potassium channels (Kv) are homotetramers composed of four voltage sensors and one pore domain. Because of high-level structural flexibility, the first mammalian Kv structure, Kv1.2 at 2.9 A, has about 37% molecular mass of the transmembrane portion not resolved. In this study, by applying a novel normal-mode-based X-ray crystallographic refinement method to the original diffraction data and structural model, we established the structure of full-length Kv1.2 in its native form. This structure offers mechanistic insights into voltage sensing. Particularly, it shows a hydrophobic layer of about 10 A at the midpoint of the membrane bilayer, which is likely the molecular basis for the observed "focused electric field" of Kv1.2 between the internal and external solutions. This work also demonstrated the potential of the refinement method in bringing up large chunks of missing densities, thus beneficial to structural refinement of many difficult systems.


Subject(s)
Kv1.2 Potassium Channel/chemistry , Animals , Anisotropy , Binding Sites , Cell Membrane/metabolism , Crystallography, X-Ray/methods , Glycosylation , Hydrophobic and Hydrophilic Interactions , Ion Channels/chemistry , Lipid Bilayers/chemistry , Models, Molecular , Molecular Conformation , Protein Conformation , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/chemistry
12.
FEBS J ; 277(14): 2912-20, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20561055

ABSTRACT

Neuronal growth-inhibitory factor (GIF), also named metallothionein-3, inhibits the outgrowth of neuronal cells. Recent studies on the structure of human GIF, carried out using NMR and molecular dynamics simulation techniques, have been summarized. By studying a series of protein-engineered mutants of GIF, we showed that the bioactivity of GIF is modulated by multiple factors, including the unique TCPCP motif-induced characteristic conformation, the solvent accessibility and dynamics of the metal-thiolate cluster, and the domain-domain interactions.


Subject(s)
Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Animals , Humans , Metallothionein 3 , Models, Molecular , Protein Engineering , Structure-Activity Relationship
13.
Protein J ; 29(1): 32-43, 2010 Jan.
Article in English | MEDLINE | ID: mdl-20013305

ABSTRACT

The biological function and stability of a cytochrome P450 (CYP) mainly depend on the subtle properties of the residues in the active site cavity, which are generally more divergent among proteins than other parts of the protein. As the most unique member of human CYP2C family, CYP2C8 has an isoleucine (Ile) 476 instead of phenylalanine (Phe) in substrate recognizing site 6 (SRS6). However, the role of Ile476 of CYP2C8 is still unknown. Therefore, six site-directed mutants of CYP2C8 were constructed to better define this. By UV-visible and circular dichroism spectroscopy studies, we studied for the first time the structural stability and all-trans-retinoic acid binding capability of the CYP2C8 variants. We found that the ferric CYP2C8 went through three states during thermal unfolding. Combined with substrate binding studies, our data revealed that residue 476 was involved in contact with substrate and was important for maintaining the thermal stability of CYP2C8.


Subject(s)
Aryl Hydrocarbon Hydroxylases/chemistry , Isoleucine/chemistry , Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/metabolism , Binding Sites , Cytochrome P-450 CYP2C8 , Humans , Molecular Conformation , Protein Binding , Protein Folding , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity
14.
Acta Crystallogr D Biol Crystallogr ; 65(Pt 7): 633-43, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19564683

ABSTRACT

The structural refinement of large complexes at the lower resolution limit is often difficult and inefficient owing to the limited number of reflections and the frequently high-level structural flexibility. A new normal-mode-based X-ray crystallographic refinement method has recently been developed that enables anisotropic B-factor refinement using a drastically smaller number of thermal parameters than even isotropic refinement. Here, the method has been systematically tested on a total of eight systems in the resolution range 3.0-3.9 A. This series of tests established the most applicable scenarios for the method, the detailed procedures for its application and the degree of structural improvement. The results demonstrated substantial model improvement at the lower resolution limit, especially in cases in which other methods such as the translation-libration-screw (TLS) model were not applicable owing to the poorly converged isotropic B-factor distribution. It is expected that this normal-mode-based method will be a useful tool for structural refinement, in particular at the lower resolution limit, in the field of X-ray crystallography.


Subject(s)
Crystallography, X-Ray/methods , Proteins/chemistry , Animals , Cation Transport Proteins/chemistry , Magnesium/chemistry , Models, Molecular , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Proteins/metabolism , Thermodynamics
15.
FEBS J ; 276(13): 3547-58, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19490120

ABSTRACT

Human neuronal growth inhibitory factor (hGIF) is able to inhibit the outgrowth of neurons. As compared with the amino acid sequences of metallothionein 1/2, hGIF contains two insertions: a Thr at position 5 and an acidic hexapeptide EAAEAE(55-60) close to the C-terminus. Moreover, all mammalian growth inhibitory factor sequences contain a conserved CPCP(6-9) motif. Previous studies have demonstrated that the TCPCP(5-9) motif is pivotal to its bioactivity, but no specific role has been assigned to the unique EAAEAE(55-60) insert. To investigate the potential structural and biological significance of the EAAEAE(55-60) insert, several mutants were constructed and investigated in detail. Notably, deletion of the acidic insert (the Delta55-60 mutant) reduced the inhibitory activity, whereas the bioactivities of other mutants did not change much. Then, spectroscopic characterization and molecular dynamics simulation were performed to investigate the potential causes of the reduced bioactivity of the Delta55-60 mutant. It was found that the domain-domain interaction mechanism of hGIF was different from that of metallothionein 2. It was also shown that the acidic insert could regulate the interdomain interactions in hGIF, leading to the structural change in the beta-domain, which resulted in the alteration of the solvent accessibility and metal release ability, thus playing an important role in the biological activity of hGIF. Our studies provided useful information on the domain-domain interaction at the molecular level for the first time, and shed new light on the mechanism of the bioactivity of hGIF.


Subject(s)
Amino Acid Sequence , Conserved Sequence/genetics , Nerve Tissue Proteins , Protein Conformation , Amino Acid Sequence/genetics , Animals , Cells, Cultured , Humans , Hydrogen-Ion Concentration , Metallothionein 3 , Models, Molecular , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/cytology , Neurons/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Denaturation , Rats , Rats, Wistar , Zinc/metabolism
16.
Biometals ; 22(5): 817-26, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19306065

ABSTRACT

Metallothinein-3 (MT3), also named neuronal growth inhibitory factor (GIF), is attractive by its distinct neuronal growth inhibitory activity, which is not shared by other MT isoforms. The polypeptide chain of GIF is folded into two individual domains, which are connected by a highly conserved linker, KKS. In order to figure out the significance of the conserved segment, we constructed several mutants of human GIF (hGIF), including the K31/32A mutant, the K31/32E mutant and the KKS-SP mutant by site-directed mutagenesis. pH titration and DTNB reaction exhibited that all the three mutations made the beta-domain lower in stability and looser. More significantly, change of KKS to SP also altered the general backbone conformation and metal-thiolate cluster geometry. Notably, bioassay results showed that the bioactivity of the K31/32A mutant and the K31/32E mutant decreased obviously, while the KKS-SP mutant lost inhibitory activity completely. Based on these results, we proposed that the KKS linker was a crucial factor in modulating the stability and the solvent accessibility of the Cd(3)S(9) cluster in the beta-domain through domain-domain interactions, thus was indispensable to the biological activity of hGIF.


Subject(s)
Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Amino Acid Sequence , Animals , Cerebral Cortex/cytology , Cerebral Cortex/metabolism , Humans , Hydrogen-Ion Concentration , Male , Metallothionein 3 , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Nerve Tissue Proteins/genetics , Neurons/cytology , Protein Structure, Tertiary , Rats , Rats, Wistar , Sequence Homology, Amino Acid
17.
J Inorg Biochem ; 102(11): 1965-72, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18757100

ABSTRACT

Human metallothionein-3 (hMT3), also named as human neuronal growth inhibitory factor (hGIF), can inhibit the outgrowth of embryonic cortical neurons in the presence of brain extracts. In order to systematically study the structure-property-reactivity-function relationship of hGIF, our laboratory designed a series of mutants and studied their structure, property, reactivity and functions by a series of chemical and biological tools including UV spectroscopy, CD spectroscopy, NMR, chemical reaction and primary neuronal culture assays. In summary, we concluded that the bioactivity of hGIF was regulated by multiple factors, including the (6)CPCP(9) motif, an additional threonine insert at sequence position 5, domain-domain interactions, the structure and stability of the metal-thiolate cluster and the linker. Our studies provide more and more evidences which revealed that the bioactivity of hGIF is mainly related to the essential metal release and its characteristic conformation.


Subject(s)
Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Amino Acid Sequence , Catalytic Domain , Circular Dichroism , Conserved Sequence , Humans , Metallothionein 3 , Models, Molecular , Molecular Sequence Data , Neurons/metabolism , Protein Structure, Tertiary , Structure-Activity Relationship , Threonine/chemistry , Threonine/genetics
18.
Biochem Biophys Res Commun ; 372(4): 779-84, 2008 Aug 08.
Article in English | MEDLINE | ID: mdl-18533104

ABSTRACT

It has been reported that the (6)CPCP(9) motif near the N-terminus is pivotal to the inhibitory activity of human neuronal growth inhibitory factor (hGIF). In order to better understand the biological significance of this region on the structure, property and function of hGIF, we introduced a highly flexible residue, Gly, either in front of the (6)CPCP(9) motif (the IG6 mutant, TGCPCP) or in the middle of it (the IG8 mutant, TCPGCP) and investigated their structural and metal binding properties in detail. The results showed that the overall structure and the stability of the metal-thiolate clusters of the two mutants were comparable to that of hGIF. However, the bioassay results showed that the bioactivity of the IG6 mutant decreased significantly, while the bioactivity of the IG8 mutant was almost abolished. Molecular dynamics simulation results showed that the backbone of the IG6 mutant exhibited high similarity to that of hGIF, and the two prolines could still induce structural constraints on the (6)CPCP(9) tetrapeptide and form a similar conformation with that of hGIF, however, the conformation of the first five amino acid residues in the N-terminus was quite different. In hGIF, the five residues are twisted and form a restricted conformation, while in the IG6 mutant this peptide extends more naturally and smoothly, which is similar to that of MT2. As to the IG8 mutant, the Gly insertion broke the (6)CPCP(9) motif, thus probably abolishing the interactions with other molecules and eliminating its inhibitory activity. Based on these results, we suggested that although the structure adopted by the (6)CPCP(9) motif is the determinant factor of the inhibitory bioactivity of hGIF, other residues within the N-terminal fragment (residue 1-13) may also influence the peptide conformation and contribute to the protein's bioactivity.


Subject(s)
Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Cells, Cultured , Conserved Sequence , Glycine/chemistry , Glycine/genetics , Humans , Metallothionein 3 , Molecular Sequence Data , Mutation , Nerve Tissue Proteins/genetics , Neurons/metabolism , Protein Conformation , Rats
19.
Protein J ; 27(3): 197-203, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18066653

ABSTRACT

Apocytochrome b5 (apocyt b5), a small b-type cytochrome with heme prosthetic group removal, has been subjected to steered molecular dynamics (SMD) simulations for investigating the consequences of mechanical force-induced unfolding. Both constant velocity (0.5 and 1.0 A/ps) and constant force (500, 750 and 1000 pN) stretching have been employed to model forced unfolding of apocyt b5. The results of SMD simulations elucidate that apocyt b5 is protected against external stress mainly through the interstrand hydrogen bonding between its beta1-beta2 and beta2-beta3 strands, highlighting the importance of hydrophobic core 2 in stabilization of apocyt b5. The existence of intermediate states manifested by current simulations in the forced unfolding pathway of apocyt b5 is different from the observations in pervious thermal or chemical unfolding studies in the absence of force. The present study could thus provide insights into the relationship between the two cooperative functional modules of apocyt b5 and also guide the rational molecular design of heme proteins.


Subject(s)
Cytochromes b5/chemistry , Models, Molecular , Protein Folding , Amino Acid Sequence , Cytochromes b5/metabolism , Magnetic Resonance Spectroscopy , Microscopy, Atomic Force , Protein Conformation , Protein Transport
20.
Proteins ; 68(1): 255-66, 2007 Jul 01.
Article in English | MEDLINE | ID: mdl-17427961

ABSTRACT

The beta-domain of metallothionein-3 (MT3) has been reported to be crucial to the neuron growth inhibitory bioactivity. Little detailed three-dimensional structural information is available to present a reliable basis for elucidation on structure-property-function relationships of this unique protein by experimental techniques. So, molecular dynamics simulation is adopted to study the structure of beta-domain of MT3. In this article, a 3D structural model of beta-domain of MT3 was generated. The molecular simulations provide detailed protein structural information of MT3. As compared with MT2, we found a characteristic conformation formed in the fragment (residue 1-13) at the N-terminus of MT3 owing to the constraint induced by 5TCPCP9, in which Pro7 and Pro9 residues are on the same side of the protein, both facing outward and the two 5-member rings of prolines are arranged almost in parallel, while Thr5 is on the opposite side. Thr5 in MT3 is also found to make the first four residues relatively far from the fragment (residue 23-26) as compared with MT2. The simulated structure of beta-domain of MT3 is looser than that of MT2. The higher energy of MT3 than that of MT2 calculated supports these conclusions. Simulation on the four isomer arising from the cis- or trans-configuration of 6CPCP9 show that the trans-/trans-isomer is energetic favorable. The partially unfolding structure of beta-domain of MT3 is also simulated and the results show the influence of 6CPCP9 sequence on the correct folding of this domain. The correlations between the bioactivity of MT3 and the simulated structure as well as the folding of beta-domain of MT3 are discussed based on our simulation and previous results.


Subject(s)
Models, Molecular , Nerve Tissue Proteins/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary , Amino Acid Motifs/genetics , Animals , Biophysical Phenomena , Biophysics , Cadmium/chemistry , Computer Simulation , Metallothionein 3 , Sulfur/chemistry
SELECTION OF CITATIONS
SEARCH DETAIL
...