Bioisis

"Characterization of enzymes from Legionella pneumophila involved in reversible adenylylation of Rab1"

Experimental SAS Curve

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Experimental Mass

52,000 Da

Experimental Details for BID:  1DRRAP
Experiment ID: 74
Collected at: X33
Contributors: Shkumatov, AV ,  Mueller, MP
After the pathogenic bacterium Legionella pneumophila is phagocytosed, it injects more than 250 different proteins into the cytoplasm of host cells to evade lysosomal digestion and to replicate inside the host cell. Among these secreted proteins is the protein DrrA/SidM, which has been shown to modify Rab1, a main regulator of vesicular trafficking in eukaryotic cells, by transfer of adenosine monophosphate (AMP) to Tyr77. In addition, Legionella provides the protein SidD that hydrolytically reverses the covalent modification, suggesting a tight spatial and temporal control of Rab1 function by Legionella during infection. Small angle x-ray scattering (SAXS) experiments of DrrA allowed us to validate a tentative complex model built by combining available crystallographic data. We have established the effects of adenylylation on Rab1 interactions and properties in a quantitative way. In addition, we have characterized the kinetics of DrrA catalyzed adenylylation as well as SidD catalyzed deadenylylation towards Rab1 and have determined the nucleotide specificities of both enzymes. This study enhances our knowledge of proteins subverting Rab1 function at the LCV.
Extrapolated to zero concentration from 4 different concentrations between 1 and 10 mg/mL

Electron Pair Distribution

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       Dmax → 140 Å


Guinier Plot

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     Guinier Rg → 39.0 Å

Real Space Rg → 42.7 Å

The Guinier plot is used to estimate the radius of gyration, Rg, which is taken from the slope of a line observed at low scattering angles (typically in the range where q* Rg < 1.3). This should be in reasonable agreement with the real space Rg.


Kratky Plot

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The Kratky plot can be used to visually assess the degree of "unfoldedness" of a protein or RNA sample. The plot of a well-behaved folded protein approaches the baseline at high q values creating a parabolic shape.


PDB Model fit to SAXS Data

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The red line is the calculated SAXS profile from a PDB model scaled to the experimental SAXS curve (cyan). The two curves agree with a Chi2 of 1.2.


GASBOR Model

GASBOR result was determined with the following:

Space GroupP1
Chi0.9
NSD | RMSD1.61 
variance(NSD | RMSD)0.06 
Number of Models in Average20 
Superimposed model20 

Additional Experimental Details
Title

Characterization of enzymes from Legionella pneumophila involved in reversible adenylylation of Rab1

Description

After the pathogenic bacterium Legionella pneumophila is phagocytosed, it injects more than 250 different proteins into the cytoplasm of host cells to evade lysosomal digestion and to replicate inside the host cell. Among these secreted proteins is the protein DrrA/SidM, which has been shown to modify Rab1, a main regulator of vesicular trafficking in eukaryotic cells, by transfer of adenosine monophosphate (AMP) to Tyr77. In addition, Legionella provides the protein SidD that hydrolytically reverses the covalent modification, suggesting a tight spatial and temporal control of Rab1 function by Legionella during infection. Small angle x-ray scattering (SAXS) experiments of DrrA allowed us to validate a tentative complex model built by combining available crystallographic data. We have established the effects of adenylylation on Rab1 interactions and properties in a quantitative way. In addition, we have characterized the kinetics of DrrA catalyzed adenylylation as well as SidD catalyzed deadenylylation towards Rab1 and have determined the nucleotide specificities of both enzymes. This study enhances our knowledge of proteins subverting Rab1 function at the LCV.

Publication

Characterization of enzymes from Legionella pneumophila involved in reversible adenylylation of Rab1, Journal of Biological Chemistry (under final revision)

Contributors

Shkumatov, AV ,  Mueller, MP

Genomics and Proteomics

The experiment is composed of a single gene/ORF

Abbreviated name: DrrA

Annotation: Rab1 adenylation (AMPylation) protein

GHMSIMGRIK MSVNEEQFGS LYSDERDKPL LSPTAQKKFE EYQNKLANLS KIIRENEGNE VSPWQEWENG LRQIYKEMIY DAFDALGVEM PKDMEVHFAG SLAKAQATEY SDLDAFVIVK NDEDIKKVKP VFDALNNLCQ RIFTASNQIY PDPIGINPSR LIGTPDDLFG MLKDGMVADV EATAMSILTS KPVLPRYELG EELRDKIKQE PSFSNMVSAK KFYNKAIKDF TAPKEGAEVV SVKTHIMRPI DFMLMGLREE FNLYSEDHLS APGTIRLLRE KNLLPEEQIA RIESVYNQAM SKRFELHAEH KKEHDEMPYS DAKAMLDEVA KIRELGVQRV TRIENLENAK KLWDNANSML EKGNISGYLK AANELHKFMK EKNLKEDDLR PELSDKTISP KGYAILQSLW GAASDYSRAA ATLTESTVEP GLVSAVNKMS AFFMDCKLSP NERATPDPDF KVGKSKILVG IMQFIKDVAD PTSKIWMHNT KALMNHKIAA IQKLERSNNV NDETLESVLS SKGENLSEYL SYKEYDYLFK LLLIGDSGVG KSCLLLRFAD DTYTESYIST IGVDFKIRTI ELDGKTIKLQ IWDTAGQERF RTITSSYYRG AHGIIVVYDV TDQESYANVK QWLQEIDRYA SENVNKLLVG NKSDLTTKKV VDNTTAKEFA DSLGIPFLET SNATNVEQAF MTMAAEIKKR MYATKDEGRE HRYTASTENF KNVKEKYQQM RGDALKTEIL ADFKDKLAEA TDEQSLKQIV AELKSKDEYR ILAKGQGLTT QLLGLKTSSV SSFEKMVEET RESIKSQERQ TIKIK
categoryamino acid composition(%)
HydrophobicI(6.3) V(5.2) L(9.1) M(3.4) A(7.5) G(4.8) P(3.1)
AromaticF(3.6) W(0.9) Y(3.6)
HydrophilicR(4.2) K(9.8) E(9.2) D(6.4) Q(3.6) N(4.9) H(1.5) S(7.5) T(5.4) C(0.4)