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Calcium binding by the PKD1 domain regulates interdomain flexibility in Vibrio cholerae metalloprotease PrtV.

Edwin A, Rompikuntal P, Björn E, Stier G, Wai SN, Sauer-Eriksson AE - FEBS Open Bio (2013)

Bottom Line: The domain arrangement of PrtV is likely to play an important role in these maturation steps, which are known to be regulated by calcium.The structure reveals a previously uncharacterized Ca(2+)-binding site located near linker regions between domains.Conformational changes in the Ca(2+)-free and Ca(2+)-bound forms suggest that Ca(2+)-binding at the PKD1 domain controls domain linker flexibility, and plays an important structural role, providing stability to the PrtV protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Umeå University, Umeå SE-901 87, Sweden ; Umeå Centre for Microbial Research (UCMR), Umeå University, Umeå SE-901 87, Sweden.

ABSTRACT
Vibrio cholerae, the causative agent of cholera, releases several virulence factors including secreted proteases when it infects its host. These factors attack host cell proteins and break down tissue barriers and cellular matrix components such as collagen, laminin, fibronectin, keratin, elastin, and they induce necrotic tissue damage. The secreted protease PrtV constitutes one virulence factors of V. cholerae. It is a metalloprotease belonging to the M6 peptidase family. The protein is expressed as an inactive, multidomain, 102 kDa pre-pro-protein that undergoes several N- and C-terminal modifications after which it is secreted as an intermediate variant of 81 kDa. After secretion from the bacteria, additional proteolytic steps occur to produce the 55 kDa active M6 metalloprotease. The domain arrangement of PrtV is likely to play an important role in these maturation steps, which are known to be regulated by calcium. However, the molecular mechanism by which calcium controls proteolysis is unknown. In this study, we report the atomic resolution crystal structure of the PKD1 domain from V. cholera PrtV (residues 755-838) determined at 1.1 Å. The structure reveals a previously uncharacterized Ca(2+)-binding site located near linker regions between domains. Conformational changes in the Ca(2+)-free and Ca(2+)-bound forms suggest that Ca(2+)-binding at the PKD1 domain controls domain linker flexibility, and plays an important structural role, providing stability to the PrtV protein.

No MeSH data available.


Related in: MedlinePlus

Difference Fourier maps showing the quality of the electron density at a representative residue (Leu34). The residue is represented as a ball-and-stick model. The 2mFo-DFc electron density of the refined structure is shown by a blue mesh contoured at 1σ. The green mesh shows the mFo-DFc electron density omit map contoured at +3σ. The positions of hydrogen atoms, not included in refinement or in map calculations, are clearly indicated in the map. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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fig0002: Difference Fourier maps showing the quality of the electron density at a representative residue (Leu34). The residue is represented as a ball-and-stick model. The 2mFo-DFc electron density of the refined structure is shown by a blue mesh contoured at 1σ. The green mesh shows the mFo-DFc electron density omit map contoured at +3σ. The positions of hydrogen atoms, not included in refinement or in map calculations, are clearly indicated in the map. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Mentions: With the X-ray diffraction data from a single crystal, the structure of the 85-residue PKD1 domain (residues Glu755-Asn839) from V. cholerae metalloprotease PrtV was determined by the molecular replacement method. The asymmetric unit contained two molecules: chains A and B. Apart from a few residues at the N- and C-termini, all protein residues could be modeled into the electron density. The overall quality of the electron density was excellent with clearly defined hydrogen atoms for most of the residues (Fig. 2). Weak or no electron density was observed for the side-chains of residues Ile757, Lys766, Glu768, Met773, Gln775, Gln830, Lys834 in both chains and residue Thr837 from chain B: these residues are situated at the surface of the molecule. The final model contains residues Ile757-Pro838 of chain A, and residues Ile757-Thr837 of chain B. In both chains, several residues are modeled in multiple conformations (2 or 3). The first two visible residues, Ile757-Ala758, at the N-terminus of chain B are modeled in two conformations depending on whether or not a Ca2+ ion is bound to the main chain carbonyl oxygen of Ala758.


Calcium binding by the PKD1 domain regulates interdomain flexibility in Vibrio cholerae metalloprotease PrtV.

Edwin A, Rompikuntal P, Björn E, Stier G, Wai SN, Sauer-Eriksson AE - FEBS Open Bio (2013)

Difference Fourier maps showing the quality of the electron density at a representative residue (Leu34). The residue is represented as a ball-and-stick model. The 2mFo-DFc electron density of the refined structure is shown by a blue mesh contoured at 1σ. The green mesh shows the mFo-DFc electron density omit map contoured at +3σ. The positions of hydrogen atoms, not included in refinement or in map calculations, are clearly indicated in the map. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3722578&req=5

fig0002: Difference Fourier maps showing the quality of the electron density at a representative residue (Leu34). The residue is represented as a ball-and-stick model. The 2mFo-DFc electron density of the refined structure is shown by a blue mesh contoured at 1σ. The green mesh shows the mFo-DFc electron density omit map contoured at +3σ. The positions of hydrogen atoms, not included in refinement or in map calculations, are clearly indicated in the map. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Mentions: With the X-ray diffraction data from a single crystal, the structure of the 85-residue PKD1 domain (residues Glu755-Asn839) from V. cholerae metalloprotease PrtV was determined by the molecular replacement method. The asymmetric unit contained two molecules: chains A and B. Apart from a few residues at the N- and C-termini, all protein residues could be modeled into the electron density. The overall quality of the electron density was excellent with clearly defined hydrogen atoms for most of the residues (Fig. 2). Weak or no electron density was observed for the side-chains of residues Ile757, Lys766, Glu768, Met773, Gln775, Gln830, Lys834 in both chains and residue Thr837 from chain B: these residues are situated at the surface of the molecule. The final model contains residues Ile757-Pro838 of chain A, and residues Ile757-Thr837 of chain B. In both chains, several residues are modeled in multiple conformations (2 or 3). The first two visible residues, Ile757-Ala758, at the N-terminus of chain B are modeled in two conformations depending on whether or not a Ca2+ ion is bound to the main chain carbonyl oxygen of Ala758.

Bottom Line: The domain arrangement of PrtV is likely to play an important role in these maturation steps, which are known to be regulated by calcium.The structure reveals a previously uncharacterized Ca(2+)-binding site located near linker regions between domains.Conformational changes in the Ca(2+)-free and Ca(2+)-bound forms suggest that Ca(2+)-binding at the PKD1 domain controls domain linker flexibility, and plays an important structural role, providing stability to the PrtV protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Umeå University, Umeå SE-901 87, Sweden ; Umeå Centre for Microbial Research (UCMR), Umeå University, Umeå SE-901 87, Sweden.

ABSTRACT
Vibrio cholerae, the causative agent of cholera, releases several virulence factors including secreted proteases when it infects its host. These factors attack host cell proteins and break down tissue barriers and cellular matrix components such as collagen, laminin, fibronectin, keratin, elastin, and they induce necrotic tissue damage. The secreted protease PrtV constitutes one virulence factors of V. cholerae. It is a metalloprotease belonging to the M6 peptidase family. The protein is expressed as an inactive, multidomain, 102 kDa pre-pro-protein that undergoes several N- and C-terminal modifications after which it is secreted as an intermediate variant of 81 kDa. After secretion from the bacteria, additional proteolytic steps occur to produce the 55 kDa active M6 metalloprotease. The domain arrangement of PrtV is likely to play an important role in these maturation steps, which are known to be regulated by calcium. However, the molecular mechanism by which calcium controls proteolysis is unknown. In this study, we report the atomic resolution crystal structure of the PKD1 domain from V. cholera PrtV (residues 755-838) determined at 1.1 Å. The structure reveals a previously uncharacterized Ca(2+)-binding site located near linker regions between domains. Conformational changes in the Ca(2+)-free and Ca(2+)-bound forms suggest that Ca(2+)-binding at the PKD1 domain controls domain linker flexibility, and plays an important structural role, providing stability to the PrtV protein.

No MeSH data available.


Related in: MedlinePlus