Comparative Study of Bacterial SPOR Domains Identifies Functionally Important Differences in Glycan Binding Affinity

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• SPOR domains are bacterial protein modules that target proteins to the cell division site (divisome).
• They achieve this by binding to specific peptidoglycan (PG) structures called denuded glycans, which are generated by cell wall amidases.
• While all known SPOR domains bind denuded glycans, their relative binding affinities were previously uncharacterized.

Purpose of the Study:

• To quantify the relative peptidoglycan (PG) glycan binding affinities of different bacterial SPOR domains.
• To investigate whether variations in PG binding affinity impact the functional roles of SPOR domain-containing proteins in bacterial cell division.

Main Methods:

• Isothermal titration calorimetry (ITC) was employed to measure the dissociation constants (Kd) for PG glycan binding of five SPOR domains from *Escherichia coli* and *Cytophaga hutchinsonii*.
• Functional analyses were performed using 'swap' proteins, where the native SPOR domains of *E. coli* DamX and FtsN were replaced with heterologous SPOR domains.

Main Results:

• The PG binding affinities (Kd) varied significantly, ranging from approximately 1 µM for *E. coli* DamX^SPOR^ and *C. hutchinsonii* CHU2221^SPOR^ to about 10 µM for *E. coli* FtsN^SPOR^.
• All tested SPOR domain swap proteins successfully localized to the bacterial division site.
• While all heterologous SPOR domains supported FtsN function in cell division, only the high-affinity CHU2221^SPOR^ domain restored normal DamX function.

Conclusions:

• Bacterial SPOR domains exhibit distinct affinities for denuded PG glycans.
• These affinity differences are functionally relevant for certain SPOR domain proteins, such as DamX, influencing their role in proper cell division.
• The findings highlight the importance of specific PG-binding affinities for the precise targeting and function of proteins within the bacterial divisome.