ATP-binding cassette transporter and heme binding protein SiaA

1
ATP-binding cassette transporter and heme binding
protein SiaA

• Yau Fong Chan (Kyle)
• Chem8360

2

• Intorduction of ABC transporter
• BtuCDF complex and mechanism
• Streptococcus pyogenes. Study of SiaA and related
  experiment

3
ATP-binding cassette transporter

• Serve as channels to transport molecules across
  cell membranes.
• Facilitate the import of nutrients into cells
  or export toxic products into the surrounding
  medium, which are essential for cellular
  homeostasis, cell growth, cell divisions, and
  bacterial immunity.
• Hydrolyze ATP and use it to move molecules
  against the concentration gradient or transport
  substrates across lipid membranes.
• Multi-domain structures that are comprised of
  two a-helical transmembrane domains (TMDs) and
  two nucleotide-binding domains (NBDs).
• Understanding ABC transporters structure and
  mechanism may help design agents to control their
  function, which might be used to treat cancer and
  drug-resistant bacteria.

4
ATP-binding cassette transporter

• ABC transporters transporter molecules such
  as
• -ions, sugars, amino acids,
  vitamins, peptides, polysaccharides, hormones,
  lipids.
• ABC transporters are involved in diverse
  cellular processes such as
• - maintenance of osmotic homeostasis,
• - nutrient uptake,
• - antigen processing,
• - cell division,
• - bacterial immunity,
• - pathogenesis

5
ATP-binding cassette transporter

• The TMDs contain multiple hydrophobic segments,
  which form the transmembrane (TM) channel.
• The primary sequences of TMDs are different from
  the NBDs,

http//www-dsv.cea.fr/art/images/devm_lems/abc3d.g
if
6
ATP-binding cassette transporter

• NBDs contain the highly conserved Walker A and B
  consensus motifs for
• nucleotide binding and the LSGGQ motif.
• TM channel contain the substrate binding sites.

http//www.biologie.uni-regensburg.de/Mikrobio/Tho
mm/Buttons/reg_fig1.gif
7
nucleotide-binding domains

• Three-dimensional structure of NBD.
• B-strands are numbered and depicted as arrows and
  a-helices as coiled ribbons.
• ATP is shown in stick form with carbon (yellow),
  nitrogen (blue), oxygen (orange) and phosphorous
  (magenta).
• Regions proposed to form interfaces with the TMDs
  are labelled IR1 and IR2.

8
BtuCDF complex structure and transport mechaism
9
BtuCD Structure and Mechanism

• BtuCD is the vitamin B12 importer of E.coli.
• The crystal structure of BtuCD provides the high
  resolution visualization of an ABC transporter.
• Bacterial ABC transporters use peri-plasmic
  binding proteins (PBPs) to capture substrate and
  present it to the membrane translocator units.
• BtuF is an example of PBPs.
• BtuF deliver vitamin B12 to BtuCD

10
BtuCD Structure and Mechanism

• In BtuCD, two copies of BtuC subunit and two
  BtuD subunits assemble to form the functional
  heterotetramer, the BtuC2D2.
• BtuCD structure revealed three features for ABC
  transporter function
• the translocation pathway
• the arrangement of ABC domains
• the transmission interface.

11
Translocation Pathway of BtuCD

• Present at interface of the two BtuC subunits
• large cavity that opens to the peri-plasmic
  space.
• Each BtuC subunits traverses the membrane 10
  times
• 20 transmembrane helices in the transporter.
• The interface between the two BtuC subunits is
  formed by anti-parallel packing of two pairs of
  helices.
• It has no structural resemblance to the binding
  pockets of vitamin B12-dependent enzyme.

12
Arrangement of ABC Domain

• The BtuD subunits are aligned such that two ATP
  hydrolysis sites are formed at the dimer
  interface between the ABC signature mofit and the
  P-loop of the other.
• Nucleotide binding and hydrolysis is due to the
  specific structure dimer instead of an individual
  domain.
• Physiological dimers could only be trapped under
  ATP-EDTA condition.
• BtuCs keep the BtuDs properly aligned.

13
Transmission- BtuC-BtuD interface region

• Architecturally conserved in ABC transporter
• Q loop of BtuD involved in the interface with
  BtuC.
• Act as a alpha-phosphate sensor that may change
  its conformation upon nucleotide binding and
  hydrolysis.
• When the binding protein BtuF has docked with
  BtuC, it signals to the ATP hydrolysis sites to
  initiate ATP hydrolysis.
• The mutation of residues located at the
  transmission interface either Interferes with
  protein folding or assembly, or affects the
  coupling of ATP hydrolysis to substrate
  translocation.
• For example, point mutation of Arg659 in
  transporter severely affects coupling.

14
BtuF structure

• BtuF is an peri-plasmic binding protein capture
  substrates, vitamin B12 and deliver them to
  BtuCD, ABC transporters.
• They are selective and have high affinity for
  their substrates.
• BtuF comprises two lobes separated by a deep
  cleft that harbors the substrate binding site.

15
Schematic Transport Mechanism of BtuC2D2F
proposed by Kaspar P Locher

• BtuF serves as an additional gate that prevents
  the escape of substrate on the wrong side of the
  membrane
• In the initial state, the central cavity of the
  translocation pathway is accessible to the
  peri-plasm and closed to the cytoplasm, and the
  two ABC domains have a relatively small shared
  interface.
• When BtuF with bound vitamin B12 is dock on the
  BtuC2D2 complex, a signal will be send across the
  membrane and triggers ATP hydrolysis.

16
Schematic Transport Mechanism of BtuC2D2F
proposed by Kaspar P Locher

• ATP hydrolysis force the cytoplasmic gate open,
  which allowing vitamin B12 escape into the
  cytoplasm.
• At the same time, the new conformation makes the
  vitamin B12 binding protein BtuF detach from
  BtuC2D2 complex, and returns to the resting
  state.

17
Streptococcus pyogenes and the SiaA Protein
18
Streptococcus pyogenes

• S. pyogenes exhibit beta hemolysis on blood
  agar.
• They colonize in the respiratory tract, blood
  stream, or skin.
• Utilizes heme of human host from hemoglobin,
  myoglobin and hemopexin hemoglobin complexes
• Cause strep throat, toxic shock syndrome,
  necrotizing fasciitis (flesh eating)
• Little information on heme uptake mechanism in S.
  pyogenes.
• Control heme uptake might allow control of
  virulent infections of S.pyogenes.

19
Streptococcus pyogenes and the SiaA Protein

• SiaA is a heme binding protein of ABC system in
  Streptococcus pyogenes.
• SiaA also serves as initial studies of the heme
  binding pocket through homology modeling and site
  directed mutagenesis.
• Targeting a heme transport protein, for example,
  SiaA, in bacterial ATP binding cassette systems
  may be advantageous, as mammalian ABC systems
  only export, while bacterial ABCs both import and
  export solutes.

20
Expression and Purification of SiaA
21
SiaA Enterokinase cleavage
22
Strong ion exchange FPLC
23
SiaA Enterokinase cleavage

• EK is use to remove the 6 x His tag from the
  N-terminal
• The mass spectrometry (MALDI) showed the observed
  mass of protein fragment is smaller than the
  predicted mass ( 280kD less than the predicted
  fragment).
• It suggested that EK cleavage happened after Arg
  instead of Lys.

24
SiaA Enterokinase cleavage

• Cleavage site is after DR instead of DK.
  Suggested that enterokinase is cleaving two
  residues beyond its normal recognition site.
• Arg and Lys are positively charged amino acid

25
BtuF and SiaA

• BtuF has high proportion of alpha helix sequence
• Heme persumably fits on edge in the central
  pocket. It can be seen that Tyr, His, and Met/His
  are all possible axial ligands.
• See if SiaA can fit into these models.

26
Showed the SiaA is predominately alpha helical.
This is expected its homology to BtuF.
27
Conclusion

• ABC transporters
• serve as channel to transport molecules across
  cell membrane and it is essential for cellular
  homeostasis and cell growth.
• hydrolyzes ATP for its conformational change.
• composed of two TMD and two NBD.
• PBP is use to capture substrate and present it to
  ABC transporter
• BtuF and SiaA are the examples of PBP
• SiaA is a heme binding protein in gram positive
  bacteria Streptococcus pyogene.
• Biophysical study of SiaA showed
• SiaA is largely alpha helical, as expected from
  its homology BtuF.
• Homology modeling of SiaA based on the homologous
  Btu and FhuD proteins allowed us to postulate
  that His, Met and Tyr are possible axial ligands.
• Mass spec strongly suggests that enterokinase is
  cleaving two residue beyond its normal
  recognition site.

28
Reference

• Locher, Strucutre and Mechanism of ABC
  transporters, Current opinion of Structural
  Biology, 2004,14-426,
• Jones, The ABC transporter structure and
  mechanism perspective on resent research, CMLS,
  Cell. Mol. Life. Sci, 2004, 61-682-699, Jones
• Locher, ABC transporter architecture and
  mechanism implications from the crystal
  structures of BtuCD and BtuF FEBS letters, 2004,
  264-268, Locher
• Liu, Heme transfer from streptococcal cell
  surface protein Shp to HisA of transporter
  HtsABC, Infect. Immun. 73, 5086-5092