Transcript Critical Evaluation of a Structural Biology Paper

Example: Critical Evaluation of a Paper
Barthe et al. (2009) Dynamic and Structural Characterization of a Bacterial FHA
Protein Reveals a New Autoinhibition Mechanism. Structure, 17:568-578
Major Observations & Conclusions:
• OdhI protein is key regulator of the TCA cycle
• Unphosphorylated form of OdhI inhibits the OdhA protein
contains FHA domain highly specific to phosphothreonine
 Odhl FHA domain required to bind OdhA, but OdHA phosphorylation is unknown

• Phosphorylated form of Odhl is inactive
conformational change between phosphorylated and unphosphorylated form
 binding of the phosphorylated N-terminal part of the protein to its own FHA domain
 new autoinhibition mechanism

Example: Critical Evaluation of a Paper
Major Results:
• Identified the CgPknA or CgPknB phosphorylated site(s) of Odhl by MS as Thr15
y1-y4 expected mass for the sequence
 y5-y10 all masses are less by 98 Da, loss of phosphate group

• Prior identification of CgPknG phosphorylation site of Odhl was Thr14.
Example: Critical Evaluation of a Paper
Major Results:
• determined NMR structure of unphosphorylated and phosphorylated form of Odhl
• Phosphorylation induces folding of N-terminal region
short amphipathic helix forms A20 to S29 faces Y11 and P116
 residues V12 to F18 bind to FHA phosphopeptide surface
 Interaction between pT15 and R87, R72 in b3/b4 loop and Asn107

• Global fold of FHA domain, a b sandwich composed of 11 strands is well conserved
b3/b4 loop adopt incompatible conformation for phosphopeptide binding in
unphosphorylated form

Example: Critical Evaluation of a Paper
Major Results:
• N-terminal domain of unphosphorylated form is unstructured and dynamic
• phosphorylated form exhibits a decrease in disorder and mobility
Example: Critical Evaluation of a Paper
Major Results:
• Large chemical shift changes consistent with pT15
• Chemical shift changes consistent with ordering of N-terminal region
• Chemical shift changes consistent with phosphopeptide binding site
Example: Critical Evaluation of a Paper
Major Conclusions Hypothesis:
• Phosphorylation at Thr14 by CgPknG could result in a similar inactive Odhl structure
• Control of OdhA activity occurs through phosphorylation of both Odhl and OdhA
• Could not obtain a structure with pT14
• Different phosphorylation sites may resulted from different signaling pathways and
stimuli
Questions:
• The importance of the binding of the N-terminal domain to the FHA domain is
dependent on the phosphorylation of OdhA. If the phosphorylation of OdhA doesn’t
occur, then the conformation change and inferred autoinhibition mechanism is not
biologically relevant.
•Does binding to OdhA occur through the N-terminal domain or FHA domain?
Is N-terminal truncation still active?
 Is it the phosphorylation of N-terminal domain or the domain binding to the FHA domain
that inactivates Odhl?

Example: Critical Evaluation of a Paper
MW = 609
Questions:
MW = 609?
• authors claim Thr15 is phosphorylated
all masses are less by 98 Da, loss of phosphate group  NOT TRUE -18 Da
 Only evidence is DY5, if Thr 14 is the phosphorylation site then Y5 would occur at 609
 Assigned DY5 peak is weak ~ 3 S/N similar intense peak next to it. Is this 609 peak?
y5-y10
Example: Critical Evaluation of a Paper
Questions:
• Why are only 1H-15N NOEs measured?
• Why not measure T1, T2 and order parameters (S2)?
Example: Critical Evaluation of a Paper
Questions:
• Will the phosphorylated residue or residues next to the phosphorylated site incur the
greatest chemical shift change?
• Is it T14 or T15?