User:Caleb Holaway/Sandbox 1: Difference between revisions

Periplasmic binding proteins (PBPs) are non-enzymatic receptors that bacteria use to sense small molecules such as carbohydrates, amino acids, and ions, and transport them into the cytoplasm.  These sorts of proteins are ubiquitous in both gram-negative and gram-positive bacteria, appearing in gram-positive bacteria as membrane-anchored lipoproteins.  The glucose/galactose binding protein (<scene name='84/842887/Gbbp/1'>GBBP</scene>) of E. Coli is amongst the best studied of these proteins.    These proteins typically exhibit a “Venus fly-trap” appearance, consisting of two globular domains connected by a small hinge region.  The hinge-like appearance is evident in GBBP.  These proteins often also work in conjunction with an ABC-binding cassette transporter which catalyzes the movement of the substance at hand across the cytoplasmic membrane.

Researchers have elucidated a few other heme binding PBPs which are functionally similar to DppA of Mtb.   <scene name='84/842887/Shut/1'>ShuT</scene> of Shigella dysenteriae and PhuT of P. aeruginosa were among the earliest of these proteins to be elucidated in 2007.  A general mechanism has been proposed for the activity of these proteins, but these proteins differ significantly structurally from DppA, so it is unlikely the specific mechanism of these proteins relates to DppA.   

A <scene name='84/842887/Dppa/4'>flexible α-helical hinge</scene> connects the <scene name='84/842887/Dppa/8'>N-terminal half (residues 1-249)</scene> to the <scene name='84/842887/Dppa/6'>C-terminal half (residues 267-541)</scene>.  This is speculated to function similar to a “clothespin spring,” maintaining a closed conformation.

Between the two halves of the protein, buried inside the core, is a <scene name='84/842887/Tetrapeptide/1'>tetrapeptide composed of Ser-Ser-Val-Thr</scene>.  The function of this is not as of yet fully understood.  The highly conserved residues <scene name='84/842887/Tetrapeptide2/1'>W442 and D445</scene> in the peptide-binding pocket of DppA were mutated to alanine by researchers. E. Coli did not yield any D445 mutant protein, suggesting it did not fold and subsequently degraded.  E. Coli did yield W442 mutant which, under spectroscopic analysis, appeared to bind and rapidly dissociate from heme.  This suggests that this residue perhaps plays a role in maintaining a specific flexibility of the DppA halves.

The CASTp software computed a solvent-accessible pocket on the closed conformation of the protein with a Richards’ solvent-accessible volume of 268Å6.  Though this contains a few heme-binding residues, including <scene name='84/842887/Tetrapeptide3/1'>His131 and Arg 179</scene>, it is too small to accommodate heme.  Normal mode analysis, though, showed that the first three lowest frequency modes produced a wide opening in the cleft which was brought on by a clamp-like, 10.7Å motion of the two halves, during which the halves slightly twisted in opposite directions.  The generated pocket has a Richards’ solvent-accessible volume ~2583Å, which would be compatible with heme binding.  Similarly, this conformation would place heme in bonding distance with several DppA residues such as <scene name='84/842887/Tetrapeptide3/2'>R179, H131, S134, E481, and L477</scene>.

Amino acids <scene name='84/842887/Tetrapeptide3/1'>His131 and Arg 179</scene> have been mutated experimentally to assess the effects of these specific residues on heme binding.  H131 mutations resulted in a nonfunctional protein which would not fold.  R179 mutations resulted in a crystalized protein nearly structurally identical to wild type DppA, with RMSD ~.11Å.  After introduction to heme, spectroscopic findings showed that heme-binding abilities of the protein were abolished in the mutant, suggesting this residue plays a significant function in binding with heme.