Using fluorescence polarization to study penicillin binding to PBP5

This article and associated images are based on a poster originally authored by Molly Jackson, Meng S. Choy, Wolfgang Peti and Suzanne Purseglove and presented at ELRIG Drug Discovery 2025 in affiliation with BMG LABTECH and University of Connecticut.

This poster is being hosted on this website in its raw form, without modifications. It has not undergone peer review but has been reviewed to meet AZoNetwork's editorial quality standards. The information contained is for informational purposes only and should not be considered validated by independent peer assessment.

Introduction

^{Benzylpenicillin was first discovered back in the 1920s, and since then, thousands of new penicillin derivatives have been discovered.1}

Penicillin is part of a class of antibiotics called β-lactams.² β-lactams are the most commonly used antibiotics. New classes of β-lactams were expanded to cover a larger range of bacterial species or combat antibiotic resistance mechanisms.¹ 

β-lactams bind to and target proteins found in the bacterial cell wall called penicillin binding proteins (PBPs).² PBPs are enzymes that are involved in the last step of the peptidoglycan cross-linking in the bacterial cell wall.¹ β-lactams inhibit the cross-linking and disrupt the building of the bacterial cell wall.

The bacterial cell wall is important for cell structure, osmosis, and protection of the organism. Once this process is disrupted, the bacteria can no longer maintain their integrity.³

Each species of bacteria contains their own particular set of PBP enzymes, and each species can have between three to eight PBP enzymes.¹ Resistance to β-lactams is a growing problem and is mainly the result of β-lactamase enzymes produced by bacteria. β-lactamase enzymes hydrolyze the β-lactams ring, rendering the antibiotic inactive.¹ 

It is important to study different variants of PBPs to understand their molecular basis and binding to β-lactams.

Enterococcus faecium is known to cause severe infections and has high resistance to β-lactams. Clinical-derived resistant PBP5 variants of E. faecium have been isolated.⁴ Interestingly, these mutations do not impact the PBP5 protein structures.² In this work, we used fluorescence polarization to determine the binding affinity of penicillin to these clinically derived resistant PBP5 variants.

Assay principle

BOCILLIN FL is a fluorescent derivative of penicillin (BODIPY-labelled penicillin V). As illustrated in Figure 1, mixing of a PBP5 with BOCILLIN FL results in an increase in FP signal over time if a binding event occurs.

Fig 1. Kinetic BOCILLIN FL fluorescence polarization assay. Image Credit: Image courtesy of Molly Jackson et al., in partnership with ELRIG (UK) Ltd.

Materials & methods

• Black, 384-well, polystyrene plates (Corning, #4511)
• BOCILLIN FL Penicillin (Thermo Scientific, #B13233)
• ThermalSeal RTS, silicone adhesive film (EXCEL Scientific, #TSS-RTQ-100)
• CLARIOstar^{® Plus} (BMG LABTECH)

Experimental procedure

WT and variant PBP5 proteins were expressed and purified from isolated Enterococcus faecium variants as previously described in Hunashal, Y. et al.². BOCILLIN FL was prepared fresh from a 1 mM stock in DMSO and used at 30 nM final concentration in the assay.

For the assay, the sample was diluted in assay buffer (100 mM sodium phosphate, pH 7.0; 0.01 % Triton X-100) and transferred to a black 384-well plate with 5 μL/well. The assay was initiated by adding 15 μL of the indicated PBP5 per well (at a final concentration of 3.6 μM) and sealing the wells with ThermalSeal. Reactions without PBP5 included only assay buffer and served as a negative control.

Instrument Settings. Source: ELRIG (UK) Ltd.

Results & discussion

Figure 2 shows wildtype PBP5 (WT, defined as PBP5 T485) binding to BOCILLIN over time, visible through the increase in the FP value (green curve). As expected, the catalytically inactive PBP5 S422A variant shows no binding (blue curve), as β-lactams strictly require the active site serine for binding.

One of the clinically most resistant PBP5 variants (T485A) binds to BOCILLIN at a much slower rate (red curve), nearly similar to the PBP5 S422A variant. Conversely, the least resistant PBP5 variant (T485M) binds to BOCILLIN at a higher rate (orange curve).

Fig 2. BOCILLIN fluorescence polarization over time with WT, S422A, T485A, and T485M PBP5 variants. Image Credit: Image courtesy of Molly Jackson et al., in partnership with ELRIG (UK) Ltd.

Conclusion

The fluorescence polarization data show that β-lactams bind strictly to the PBP5 active site via S422. Clinical PBP5 variants show a large change in fluorescence polarization, correlating with their clinical activity. The CLARIOstar ^Plus provides strong support in determining the interaction differences between the variants by monitoring the FP increase with very high sensitivity. The incubation function allows the optimum temperature to be set and maintained during the kinetic incubation study.

References

1. Bush, K. and Bradford, P.A. (2016). β-Lactams and β-Lactamase Inhibitors: An Overview. Cold Spring Harbor Perspectives in Medicine, 6(8), p.a025247. DOI: 10.1101/cshperspect.a025247. https://perspectivesinmedicine.cshlp.org/content/6/8/a025247.
2. Hunashal, Y., et al. (2023). Molecular basis of β-lactam antibiotic resistance of ESKAPE bacterium E. faecium Penicillin Binding Protein PBP5. Nature Communications, [online] 14(1), p.4268. DOI: 10.1038/s41467-023-39966-5. https://www.nature.com/articles/s41467-023-39966-5.
3. Yip, D., Gerriets, V., Penicillin. StatPearls Publishing. (2023)
4. Rice, L.B., et al. (2004). Impact of Specific pbp5 Mutations on Expression of β-Lactam Resistance in Enterococcus faecium. Antimicrobial Agents and Chemotherapy, 48(8), pp.3028–3032. DOI: 10.1128/aac.48.8.3028-3032.2004. https://journals.asm.org/doi/10.1128/aac.48.8.3028-3032.2004.

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Last Updated: Dec 12, 2025