PhosphoThr Peptide Binding Globally Rigidifies Much of the FHA Domain from Arabidopsis Receptor Kinase-Associated Protein Phosphatase^,

Abstract

A net increase in the backbone rigidity of the kinase-interacting FHA domain (KI-FHA) from the Arabidopsis receptor kinase-associated protein phosphatase (KAPP) accompanies the binding of a phosphoThr peptide from its CLV1 receptor-like kinase partner, according to 15 N NMR relaxation at 11.7 and 14.1 T. All of the loops of free KI-FHA display evidence of nsec-scale motions. Many of these same residues have residual dipolar couplings that deviate from structural predictions. Binding of the CLV1 pT868 peptide seems to reduce nsec-scale fluctuations of all loops, including half of the residues of recognition loops. Residues important for affinity are found to be rigid, i.e. conserved residues and residues of the subsite for the key pT+3 peptide position. -This behavior parallels SH2 and PTB domain recognition of pTyr peptides. PhosphoThr peptide binding increases KI-FHA backbone rigidity (S 2 ) of three recognition loops, a loop nearby, seven strands from the β-sandwich, and a distal loop. Compensating the trend of increased rigidity, binding enhances fast mobility at a few sites in four loops on the periphery of the recognition surface and in two loops on the far side of the β-sandwich. Line broadening evidence of µsec to msec-scale fluctuations occurs across the six-stranded β-sheet and nearby edges of the β-sandwich; this forms a network connected by packing of interior side chains and H-bonding. A patch of the slowly fluctuating residues coincides with the site of segment-swapped dimerization in crystals of the FHA domain of human Chfr. Phosphopeptide binding introduces µsec to msec-scale fluctuations to more residues of the long 8/9 *To whom correspondence should be addressed, E-mail: vandorens@missouri.edu, Phone: 1 (573) 882-5113, FAX: 1 (573) 884-4812. † These authors contributed equally to this work. ‡ Current address: Department of Chemistry, 225 Prospect St., Yale University, New Haven, CT 06520The NMR relaxation data and their model-free interpretation are available for free and bound KI-FHA under BMRB accession codes 5841 and 6474 at www.bmrb.wisc.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 January 29. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript recognition loop of KI-FHA. The rigidity of this FHA domain appears to couple as a whole to pThr peptide binding. KeywordsFHA domain; phosphoThr-binding module; protein-protein interactions; NMR relaxation; relaxation dispersion; dynamics; residual dipolar couplings FHA domains bind phosphothreonine-and phosphoserine-containing partners in diverse eukaryotic signaling pathways that include DNA damage repair, cell proliferation, pre-mRNA splicing, forkhead transcription factors, Ring-finger proteins, and kinesins (1). Several highresolution structures of FHA domains have appeared (2-8), but no detailed studies of their dynamics. Flexibility often appears to correlate with binding events, including affinity of protein modules for peptides (9-11). Residue-specific effects on...

Mechanistic Insights into Phosphoprotein-Binding FHA Domains

“…The average rigidity of the backbone on the fast timescale is increased with pThr peptide bound 26. In fact, sites of binding-enhanced rigidity are observed with the β-sandwich in all four of the β-hairpins (those with blue coloration in Figure 5) 26. The nanosecond fluctuations enriched in its loops are quenched once the phosphopeptide is bound.…”

“…This swaps strands 9–11 into a β-sandwich with strands 1–7 from the other chain. In KI-FHA, β-strand 8 lacks measurable structural stability 32 and is mobile on the millisec scale 26. If low stability and mobility of β-strand 8 generalize to other FHA domains, it could account for β-strand 8 being malleable enough to straighten out in crystals of segment-swapped CHFr.…”

“…The nanosecond fluctuations enriched in its loops are quenched once the phosphopeptide is bound. Perhaps the binding-increased rigidity of KI-FHA reflects the favorable enthalpy of association 18 that might result from improved internal packing and hydrogen bonding 26.…”

Partially Unfolded Forms and Non-two-state Folding of a β-Sandwich: FHA Domain from Arabidopsis Receptor Kinase-associated Protein Phosphatase

“…In the structure, β2 can instead be grouped with β3 to form a green foldon since their side chains pack together in the interior. β2's lower sensitivity to denaturant than that of β1 seems to correlate with (i) the greater surface accessibility of 24% for β2 than the 11% of β1 and (ii) slightly greater flexibility in β2 (S 2 = 0.846 for His196) than in β1 (S 2 of 0.914) (Ding et al, 2005). The NHX results on OspA show that some of the strands on the edges of its β-sheets (β-strands 1, 4 and 21 for example) also have lower m-values and stability than their neighbors to the interior of the sheet 21 .…”

Crystal Structure of the Pml1p Subunit of the Yeast Precursor mRNA Retention and Splicing Complex

“…23 Similarly, loop regions in the FHA domain of Ki67 have been shown to adopt a fixed structure in the presence of a binding partner. 15 NMR chemical shift changes have indicated a similar situation in NIPP1, where several loops are restructured upon phosphopeptide binding.…”