Dataset Information

Crystal Structure of the SH3 Domain of ASAP1 in Complex with the Proline Rich Motif (PRM) of MICAL1 Reveals a Unique SH3/PRM Interaction Mode.

ABSTRACT: SH3 domains are common protein binding modules. The target sequence of SH3 domains is usually a proline-rich motif (PRM) containing a minimal "PxxP" sequence. The mechanism of how different SH3 domains specifically choose their targets from vast PxxP-containing sequences is still not very clear, as many reported SH3/PRM interactions are weak and promiscuous. Here, we identified the binding of the SH3 domain of ASAP1 to the PRM of MICAL1 with a sub-μM binding affinity, and determined the crystal structure of ASAP1-SH3 and MICAL1-PRM complex. Our structural and biochemical analyses revealed that the target-binding pocket of ASAP1-SH3 contains two negatively charged patches to recognize the "xPx + Px+" sequence in MICAL1-PRM and consequently strengthen the interaction, differing from the typical SH3/PRM interaction. This unique PRM-binding pocket is also found in the SH3 domains of GTPase Regulator associated with focal adhesion kinase (GRAF) and Src kinase associated phosphoprotein 1 (SKAP1), which we named SH3^AGS. In addition, we searched the Swiss-Prot database and found ~130 proteins with the SH3^AGS-binding PRM in silico. Finally, gene ontology analysis suggests that the strong interaction between the SH3^AGS-containing proteins and their targets may play roles in actin cytoskeleton regulation and vesicle trafficking.

SUBMITTER: Jia X

PROVIDER: S-EPMC9865144 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Crystal Structure of the SH3 Domain of ASAP1 in Complex with the Proline Rich Motif (PRM) of MICAL1 Reveals a Unique SH3/PRM Interaction Mode.

Jia Xuanyan X Lin Leishu L Xu Shun S Li Lingxuan L Wei Zhiyi Z Yu Cong C Niu Fengfeng F

International journal of molecular sciences 20230111 2

SH3 domains are common protein binding modules. The target sequence of SH3 domains is usually a proline-rich motif (PRM) containing a minimal "PxxP" sequence. The mechanism of how different SH3 domains specifically choose their targets from vast PxxP-containing sequences is still not very clear, as many reported SH3/PRM interactions are weak and promiscuous. Here, we identified the binding of the SH3 domain of ASAP1 to the PRM of MICAL1 with a sub-μM binding affinity, and determined the crystal ...[more]

PMID: 36674928

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Project description:An eight-amino-acid synthetic peptide (IIe1-Phe2-Pro3-Pro4-Val5-Pro6-Gly7-Pro8) corresponding to the conserved proline-rich motif (PRM) of the intracellular domain of the prolactin receptor (PRL-R) was studied by one- and two-dimensional (1D and 2D) proton NMR spectroscopy in water and DMSO in order to characterize its conformational dynamics. The purified PRL-R PRM peptide eluted as two partially resolved peaks in equilibrium on reverse-phase HPLC (RP-HPLC) at 20 degrees C with a ratio of 60:40. At 30 degrees C, the two peaks coalesced into a single peak The two RP-HPLC peaks correspond to two peptide conformers resulting from the slow cis-trans isomerization of one of the four proline amide bonds. Although the peptide has only three amide (NH) protons, its ID NMR spectrum in water contains approximately 15 discernible NH region peaks, providing evidence for multiple conformers. The amide resonances were assigned on the basis of 2D-COSY spectra, chemical shift values resonance splitting patterns and temperature coefficients. The cis:trans ratio for each proline in water, calculated from integrated intensities and/or peak heights of the appropriate resonances, were Phe2-Pro3 (35:65), Pro3-Pro4 (40:60), Val5-Pro6 (70:30), and Gly7-Pro8 (30:70). Temperature studies (25-70 degrees C) were used to semi-quantitatively estimate the rates of isomerization for the different prolines. In water, Pro8 undergoes rapid isomerization; Pro3 isomerizes at an intermediate rate; while Pro4 and Pro6 both appear to isomerize very slowly since no coalescence of amide resonances was observed. In DMSO, only Pro4 displayed slow isomerization. Slow kinetics combined with a similar 60:40 ratio of conformers determined by RP-HPLC and NMR suggests that isomerization of the Pro3-Pro4 bond generates the two RP-HPLC peaks. Both proximal and distal proline isomerization effects were observed in NMR experiments. All of the 16 theoretical (24 = 16) proline configurations appear to exist in equilibrium in water The predominant (19%) conformation, trans3-trans4-cis6-trans8, may reflect the configuration of the PRM prolines in the native PRL-R. Isomerization of Pro6 from cis to trans generates an interaction between the peptide N-and C-termini, suggesting an overall pseudo-cyclic conformation. This all-trans proline configuration may play an important biochemical role in the function of cytokine/haematopoietin receptors. A model is proposed which suggests that isomerization of the PRM by an immunophilin such as the FK 506-binding protein (FKBP) serves as an on-off switch for cytokine receptor activation.

Dataset Information

Crystal Structure of the SH3 Domain of ASAP1 in Complex with the Proline Rich Motif (PRM) of MICAL1 Reveals a Unique SH3/PRM Interaction Mode.

Publications

Crystal Structure of the SH3 Domain of ASAP1 in Complex with the Proline Rich Motif (PRM) of MICAL1 Reveals a Unique SH3/PRM Interaction Mode.

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