DI1000316 Autoinhibitory region bound to p21-activated kinase 4 (PAK4) kinase domain 4l67 X-ray 2.80 Homo sapiens Inferred from homology 23876315 Wang W, Lim L, Baskaran Y, Manser E, Song J Biochem. Biophys. Res. Commun. 2013 Six human PAK members are classified into groups I (PAKs 1-3) and II (PAK4-6). Previously, only group I PAKs were thought to be auto-inhibited but very recently PAK4, the prototype of group II PAKs, has also been shown to be auto-inhibited by its N-terminal regulatory domain. However, the complete auto-inhibitory domain (AID) sequence remains undefined and the mechanism underlying its auto-inhibition is largely elusive. Here, the N-terminal regulatory domain of PAK4 sufficient for auto-inhibiting and binding Cdc42/Rac was characterized to be intrinsically unstructured, but nevertheless we identified the entire AID sequence by NMR. Strikingly, an AID peptide was derived by deleting the binding-unnecessary residues, which has a Kd of 320nM to the PAK4 catalytic domain. Consequently, the PAK4 crystal structure complexed with the entire AID has been determined, which reveals that the complete kinase cleft is occupied by 20 AID residues composed of an N-terminal α-helix and a previously-identified pseudosubstrate motif, thus achieving auto-inhibition. Our study reveals that PAK4 is auto-inhibited by a novel mechanism which is completely different from that for PAK1, thus bearing critical implications for design of inhibitors specific for group II PAKs. GO:0004674 protein serine/threonine kinase activity GO:0005524 ATP binding GO:0048365 Rac GTPase binding GO:0098641 cadherin binding involved in cell-cell adhesion GO:0006915 apoptotic process GO:0007049 cell cycle GO:0007266 Rho protein signal transduction GO:0007346 regulation of mitotic cell cycle GO:0008283 cell proliferation GO:0016049 cell growth GO:0016477 cell migration GO:0023014 signal transduction by protein phosphorylation GO:0030036 actin cytoskeleton organization GO:0031098 stress-activated protein kinase signaling cascade GO:0032147 activation of protein kinase activity GO:0042981 regulation of apoptotic process GO:0043408 regulation of MAPK cascade GO:0060996 dendritic spine development GO:0071407 cellular response to organic cyclic compound GO:0098609 cell-cell adhesion GO:0005794 Golgi apparatus GO:0005913 cell-cell adherens junction GO:0005925 focal adhesion No modifications of the original PDB file. 2 Protein kinase B Serine/threonine-protein kinase PAK 4 Disordered Homo sapiens PRQWQSLIEESARRPKPLVDPACIT 25 O96013 36 60 4.2% UniRef90_O96013 36 60 secondary structure helix 42 45 pfam PF00786.25 PBD 10 66 A Serine/threonine-protein kinase PAK 4 Ordered Homo sapiens SHEQFRAALQLVVDPGDPRSYLDNFIKIGEGSTGIVCIATVRSSGKLVAVKKMDLRKQQRRELLFNEVVIMRDYQHENVVEMYNSYLVGDELWVVMEFLEGGALTDIVTHTRMNEEQIAAVCLAVLQALSVLHAQGVIHRDIKSDSILLTHDGRVKLSDFGFCAQVSKEVPRRKSLVGTPYWMAPELISRLPYGPEVDIWSLGIMVIEMVDGEPPYFNEPPLKAMKMIRDNLPPRLKNLHKVSPSLKGFLDRLLVRDPAQRATAAELLKHPFLAKAGPPASIVPLMRQNRTR 292 O96013 300 591 49.4% UniRef90_O96013 300 591 secondary structure helix 301 309 secondary structure helix 317 319 secondary structure beta 321 326 secondary structure beta 335 340 secondary structure beta 346 353 secondary structure helix 362 368 secondary structure beta 378 378 secondary structure beta 381 387 secondary structure beta 390 395 secondary structure beta 401 402 secondary structure helix 403 407 secondary structure helix 414 433 secondary structure beta 437 437 secondary structure helix 443 445 secondary structure beta 446 448 secondary structure beta 454 456 secondary structure beta 463 463 secondary structure beta 472 472 secondary structure helix 479 481 secondary structure helix 484 487 secondary structure beta 492 492 secondary structure helix 496 510 secondary structure helix 520 529 secondary structure helix 538 540 secondary structure helix 543 550 secondary structure helix 563 566 secondary structure helix 570 574 secondary structure helix 578 584 pfam PF00069.22 Pkinase 321 572 B The interacting region of PAK4 has been shown to be intrinsically disordered in homologous proteins (PMID:9660763 and PMID:15821030). The 65-123 region described in IDEAL entry IID50053 covers 60% of the binding sequence in a homologous protein. A The protein kinase domain involved in the interaction is known to adopt a stable structure in isolation (see Pfam domain PF00069). A solved monomeric structure of the domain is represented by PDB ID 1i09. 4fie 4fif 4fii