Insight into the binding properties of MEKK3 PB1 to MEK5 PB1 from its solution structure.
MEKK3 is a mitogen-activated protein kinase kinase kinase that participates in various signaling pathways. One of its functions is to activate the ERK5 signal pathway by phosphorylating and activating MEK5. MEKK3 and MEK5 each harbors a PB1 domain in the N-terminus, and they form a heterodimer via PB1-PB1 domain interaction that was reported to be indispensable to the activation of MEK5. Using NMR spectroscopy, we show here that a prolyl isomerization of the Gln38-Pro39 bond is present in MEKK3 PB1, which is the first case of structural heterogeneity within PB1 domains. We have solved the solution structures of both isomers and found a major difference between them in the Pro39 region. Residues Gly37-Leu40 form a type VIb beta-turn in the cis conformation, whereas no obvious character of beta-turn was observed in the trans conformation. Backbone dynamics studies have unraveled internal motions in the beta3/beta4-turn on a microsecond-millisecond time scale. Further investigation of its binding properties with MEK5 PB1 has demonstrated that MEKK3 PB1 binds MEK5 PB1 tightly with a Kd of about 10(-8) M. Mutagenesis analysis revealed that residues in the basic cluster of MEKK3 PB1 contributes differently to the PB1-PB1 interaction. Residues Lys 7 and Arg 5 play important roles in the interaction with MEK5 PB1. Taken together, this study provides new insights into structural details of MEKK3 PB1 and its binding properties with MEK5 PB1.
Hu
Qi
Q
Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, People's Republic of China.
Shen
Weiqun
W
Huang
Hongda
H
Liu
Jiangxin
J
Zhang
Jiahai
J
Huang
Xiaojuan
X
Wu
Jihui
J
Shi
Yunyu
Y
D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D048671:MAP Kinase Kinase 5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D048748:MAP Kinase Kinase Kinase 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011392:Proline / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D018076:DNA, Complementary; D011392:Proline; D048748:MAP Kinase Kinase Kinase 3; C482625:MAP3K3 protein, human; D048671:MAP Kinase Kinase 5; C482563:MAP2K5 protein, human