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...

The greater activity of MMP-12 than MMP-3 toward substrates from protein fibrils has been quantified. Why is MMP-12 the more active protease? We looked for behaviors associated with the higher activity of MMP-12 than MMP-3, using nuclear magnetic resonance to monitor backbone dynamics and residue-specific stabilities of their catalytic domain. The proteolytic activities are likely to play important roles in inflammatory diseases of arteries, lungs, joints, and intestines. Nuclear magnetic resonance line broadening indicates that regions surrounding the active sites of both proteases sample conformational substates within milliseconds. The more extensive line broadening in MMP-3 suggests greater sampling of conformational substates, affecting the full length of helix B and beta-strand IV forming the active site, and more remote sites. This could suggest more excursions to functionally incompetent substates. MMP-3 also has enhanced subnanosecond fluctuations in helix A, in the beta-hairpin of strands IV and V, and before and including helix C. Hydrogen exchange protection in the EX2 regime suggests that MMP-3 possesses 2.8 kcal/mol higher folding stability than MMP-12(E219A). The beta-sheet of MMP-3 appears to be stabilized still more. The higher stability of MMP-3 relative to MMP-12 coincides with the former's considerably lower proteolytic activity. This relationship is consistent with the hypothesis that enzymes often trade stability for higher activity.