Example: Critical Evaluation of a Paper

1. 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

2. 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.

3. 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

4. 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

5. 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

6. 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?

7. Example: Critical Evaluation of a Paper MW = 609 MW = 609? • Questions: • authors claim Thr15 is phosphorylated • y5-y10 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?

8. Example: Critical Evaluation of a Paper • Questions: • Why are only 1H-15N NOEs measured? • Why not measure T1, T2 and order parameters (S2)?

9. 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?