Disruption of the productive encounter complex results in dysregulation of DIAPH1 activity
Gregory G Theophall1, Lisa M S Ramirez1, Aaron Premo1
1Department of Chemistry, State University of New York at Albany, Albany, New York, USA.
Abstract:
The diaphanous-related formin, Diaphanous 1 (DIAPH1), is required for the assembly of Filamentous (F)-actin structures. DIAPH1 is an intracellular effector of the receptor for advanced glycation end products (RAGE) and contributes to RAGE signaling and effects such as increased cell migration upon RAGE stimulation. Mutations in DIAPH1, including those in the basic "RRKR" motif of its autoregulatory domain, diaphanous autoinhibitory domain (DAD), are implicated in hearing loss, macrothrombocytopenia, and cardiovascular diseases. The solution structure of the complex between the N-terminal inhibitory domain, DID, and the C-terminal DAD, resolved by NMR spectroscopy shows only transient interactions between DID and the basic motif of DAD, resembling those found in encounter complexes. Cross-linking studies placed the RRKR motif into the negatively charged cavity of DID. Neutralizing the cavity resulted in a 5-fold decrease in the binding affinity and 4-fold decrease in the association rate constant of DAD for DID, indicating that the RRKR interactions with DID form a productive encounter complex. A DIAPH1 mutant containing a neutralized RRKR binding cavity shows excessive colocalization with actin and is unresponsive to RAGE stimulation. This is the first demonstration of a specific alteration of the surfaces responsible for productive encounter complexation with implications for human pathology.
Insights
Diaphanous 1 (DIAPH1) protein interactions are crucial for actin assembly and cellular responses. Specific mutations in DIAPH1 linked to diseases disrupt these interactions, affecting cell migration and actin organization.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Diaphanous 1 (DIAPH1) is essential for Filamentous (F)-actin assembly and acts as an intracellular effector for the receptor for advanced glycation end products (RAGE).
- DIAPH1 dysfunction, particularly mutations in its diaphanous autoinhibitory domain (DAD) RRKR motif, is linked to human diseases like hearing loss and cardiovascular conditions.
- Understanding the structural basis of DIAPH1 regulation is key to deciphering its role in RAGE signaling and disease pathogenesis.
Purpose of the Study:
- To elucidate the structural interactions between the N-terminal inhibitory domain (DID) and the C-terminal diaphanous autoinhibitory domain (DAD) of DIAPH1.
- To investigate the functional significance of the basic "RRKR" motif within the DAD and its interaction with the DID.
- To determine the impact of altering these specific interaction surfaces on DIAPH1 function and RAGE signaling.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy to determine the solution structure of the DID-DAD complex.
- Cross-linking studies to map the spatial proximity of the RRKR motif within the DID cavity.
- Biochemical assays to measure binding affinity and association rate constants.
- Analysis of DIAPH1 mutant behavior in cellular contexts, including actin colocalization and response to RAGE stimulation.
Main Results:
- NMR and cross-linking revealed that the DIAPH1 RRKR motif forms a productive encounter complex within a negatively charged cavity on the DID.
- Neutralizing this cavity significantly reduced the binding affinity and association rate between DAD and DID.
- A DIAPH1 mutant with a neutralized RRKR binding site exhibited aberrant actin colocalization and impaired responsiveness to RAGE stimulation.
Conclusions:
- The interaction between the DIAPH1 RRKR motif and the DID cavity is critical for proper DIAPH1 regulation and function.
- This specific interaction interface plays a key role in DIAPH1's contribution to RAGE signaling and cellular processes like migration.
- Alterations in this interaction surface provide a molecular basis for DIAPH1-associated pathologies and highlight a potential therapeutic target.
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