Structural elements required for deamidation of RhoA by cytotoxic necrotizing factor 1

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Cytotoxic necrotizing factor 1 (CNF1) from pathogenic Escherichia coli is a virulence factor.
• CNF1 targets Rho-GTPases, specifically deamidating Gln-63 in RhoA, which is crucial for GTP hydrolysis.
• This modification leads to constitutive activation of Rho-GTPases and subsequent proteasomal degradation.

Purpose of the Study:

• To investigate the functional importance of specific loop segments at the entrance of the CNF1 active site.
• To identify the structural elements responsible for RhoA recognition and deamidation by CNF1.
• To understand the precise molecular interactions governing CNF1's enzymatic activity.

Main Methods:

• Site-directed mutagenesis was used to individually delete five loop segments (2, 6, 7, 8, and 9) within the catalytic domain of CNF1 (CNF1-C).
• In vitro deamidation assays were performed using wild-type and mutant CNF1-C with RhoA as a substrate.
• Kinetic parameters, including the specificity constant (kcat/Km), were determined for the interaction between CNF1-C and RhoA.

Main Results:

• Deletion of loops 8 and 9 significantly reduced or abolished RhoA deamidation, suggesting their role in RhoA recognition.
• Deletion of loop 7 resulted in protein folding errors, while loop 6 deletion had a minor effect on deamidation.
• Deletion of loop 2 increased RhoA deamidation 5-7 fold, indicating a conformational change upon RhoA binding.
• CNF1-C demonstrated high specificity, unable to deamidate RhoA with Asn-63, and exhibited a specificity constant of 825 ± 3 M⁻¹s⁻¹ for RhoA.

Conclusions:

• Loops 8 and 9 are critical for RhoA recognition by CNF1.
• Loop 2 appears to undergo a conformational rearrangement upon RhoA binding, influencing deamidation efficiency.
• The precise fit within the CNF1 active site pocket dictates its specificity and limits its reactivity towards RhoA.