Explain your model's assumptions, data, parameters, and results in a way that anyone could understand.




Our initial idea was centred around the use of RNAIII inhibitory peptide (RIP) to competitively inhibit RNAIII activating peptide (RAP) binding to the target of RAP (TRAP) [1]. According to various sources we found, this would downregulate the agr system, linked to autoinducing peptide (AIP) based quorum sensing (QS). Leading to reduced expression of RNAIII which studies had linked to reduced biofilm formation [2].

Prior to forming our model, we hit some blocks when searching for the DNA sequence of RAP.

  • We were unable to find any sequence for 'RAP' or 'RNAIII inhibitory peptide' across multiple gene and protein databases, including both the gene and protein database in NCBI and the protein database in UniProt.
  • The sequence we managed to find was 'Large ribosomal subunit protein uL2' which had 100% amino acid identity with the RAP sequence given in a paper by Korem et al. who generated a recombinant form of the protein to characterise RAP for the first time [3].
  • When we searched 'Large ribosomal subunit protein uL2' in both NCBI and UniProt protein databases again we found a sequence, however RAP was not listed as an alternative name.
  • This began to raise some flags, but we had begun some modelling alongside, which helped us test the likelihood that these proteins would actually interact.


An image of RAP aligned to 50S ribosomal protein L2 using the matchmaker tool in the visualisation program ChimeraX
Figure 1: (Blue) RAP best model from Alphafold 2 aligned to (orange) 50S ribosomal protein L2 using matchmaker tool in ChimeraX. Both amino acid sequences were sequenced from Staphylococcus aureus.

The Structure of RAP

  • Using the matchmaker command in ChimeraX the protein structures for RAP and 50S ribosomal protein uL2 have been superimposed using a pairwise sequence alignment and then by fitting the aligned residue pairs.
  • The RMSD (root-mean-square deviation) between the two sets of atoms is 0.243Å. For reference, a successful RMSD score is less than or equal to 1.5Å [4]. Providing confirmation that the 50S ribosomal binding protein uL2 was indeed the protein sequence that had been previously labelled as RAP.
  • The per residue confidence scores (pLDDT) for both proteins were mostly above 90, indicating a very high confidence score.
We then modelled the binding of RAP to TRAP using the Alphafold tool integrated into ChimeraX (predict function).

The Binding of RAP and TRAP

  • According to the mechanism proposed by Balaban, et al. [5] who claimed to discover RIP in 1998 [6] TRAP is the receptor for RAP. Therefore, we wanted to model the protein-protein interactions between RAP and TRAP to see how they would bind. Our initial thinking behind this was that we could then see how RIP would bind, in order to better understand the mechanism of competitive inhibition.
  • As we started this process the first red flag to appear was that the sequence for RAP was much longer than that of the receptor, TRAP.
    • RAP = 277 aa
    • TRAP = 167 aa
    Best model prediction for interaction between RAP (PAE domains in pink, green, yellow) and TRAP (PAE domain blue)
    Figure 2: Best model prediction for interaction between RAP (PAE domains in pink, green, yellow) and TRAP (PAE domain blue)

  • This could have been explained if TRAP was a multimer, but on RSCB PDB the global stoichiometry was found to be monomeric in both solution and the crystalline form.
  • PAE (predicted aligned error) is a useful tool for assessing the quality of inter-domain predictions. Where pLDDT is very useful for assessing the quality of individual domain structure predictions (see structure of RAP discussion), PAE has been used here [7].
  • PAE matrix for the interaction between domains in the RAP/TRAP complex, generated using PAE viewer
    Figure 3: PAE matrix for the interaction between domains in the RAP/TRAP complex, generated using PAE viewer [8].
  • The PAE is generally high (10-30 Å) - see Figure 3. This indicates that the relative position and orientation of RAP and TRAP is uncertain, meaning it should not be interpreted [7].
  • These results flagged to us that it is unlikely that RAP and TRAP do bind, suggesting that these systems may not function as described in the literature we had found.
  • Initially we thought this could mean TRAP was not the receptor, and that there was an uncharacterised sensor kinase - with the proposed mechanism being RAP binding to the SK leading to the phosphorylation of the HK domain in TRAP.

Returning to the Literature

The output from Alphafold2 and ChimeraX, paired with the struggles we faced when searching for the sequences initially, lead us to become sceptical of this mechanism. Upon returning to the literature, we found various contradictory papers. This discourse was summarised in a 2007 article [9]. To summarise, Balaban and Novick published the first paper on RIP in 1995. Subsequent experiments carried out in the same year found that the agr-stimulating activity which had been attributed to RAP could actually be due to the action of AIPs. This was disputed by Balaban's group in a 1998 paper. In the following years more papers were published on the subject, however the nail in the head came with a paper lead by Novick [10], who was an author on the first paper. This paper determined that Balaban's results could be due to AIP contamination rather than the action of RIP. Following this, Balaban published a sub-par response. This was followed by the article in which we found the 'Large ribosomal subunit protein uL2' DNA sequence [3], bringing us in a full circle. To summarise all the findings: inactivation of traP (the gene for TRAP) has no impact on S. aureus virulence (specifically the haemolytic activity), and disruption of TRAP had no effect on S. aureus surface protein profile [11]. Therefore, disproving Balaban's work. A few years later a paper was released reviewing these findings, and interrogating whether RIP could be effective [12]. The results showed that TRAP may function to reduce oxidative stress, and that RIP could be effective - but only with an amide group on the C-terminus. This is not possible to encode and replicate in bacteria. Therefore, we returned to idea generation - using the skills and knowledge we had gained to find a way to inhibit biofilm formation.


  1. Gov Y, Bitler A, Dell'Acqua G, Torres JV, Balaban N. RNAIII inhibiting peptide (RIP), a global inhibitor of Staphylococcus aureus pathogenesis: structure and function analysis. Peptides. 2001;22(10):1609-20.
  2. De Oliveira A, Pinheiro-Hubinger L, Pereira VC, Riboli DFM, Martins KB, Romero LC, Cunha MDLRDSD. Staphylococcal Biofilm on the Surface of Catheters: Electron Microscopy Evaluation of the Inhibition of Biofilm Growth by RNAIII Inhibiting Peptide. Antibiotics. 2021;10(7):879.
  3. Korem M, Sheoran AS, Gov Y, Tzipori S, Borovok I, Balaban N. Characterization of RAP, a quorum sensing activator of Staphylococcus aureus. FEMS Microbiology Letters. 2003;223(2):167-75.
  4. Hevener KE, Zhao W, Ball DM, Babaoglu K, Qi J, White SW, Lee RE. Validation of Molecular Docking Programs for Virtual Screening against Dihydropteroate Synthase. Journal of Chemical Information and Modeling. 2009;49(2):444-60.
  5. Balaban N, Goldkorn T, Gov Y, Hirshberg M, Koyfman N, Matthews HR, et al. Regulation of Staphylococcus aureus Pathogenesis via Target of RNAIII-activating Protein (TRAP). Journal of Biological Chemistry. 2001;276(4):2658-67.
  6. Balaban N, Goldkorn T, Nhan RT, Dang LB, Scott S, Ridgley RM, et al. Autoinducer of virulence as a target for vaccine and therapy against Staphylococcus aureus. Science. 1998;280(5362):438-40.
  7. AlphaFold Protein Structure Database. Frequently asked questions: EMBL European Bioinformatics Institute; [Internet]. Available from:
  8. Elfmann C, Stülke J. PAE viewer: a webserver for the interactive visualization of the predicted aligned error for multimer structure predictions and crosslinks. Nucleic Acids Research. 2023;51(W1):W404-W10.
  9. Ahmad S, Rahman H, Qasim M, Nawab J, Alzahrani KJ, Alsharif KF, Alzahrani FM. Staphylococcus epidermidis Pathogenesis: Interplay of icaADBC Operon and MSCRAMMs in Biofilm Formation of Isolates from Pediatric Bacteremia in Peshawar, Pakistan. Medicina. 2022;58(11):1510.
  10. Novick RP, Ross HF, Figueiredo AMS, Abramochkin G, Muir T. Activation and Inhibition of the Staphylococcal AGR System. Science. 2000;287(5452):391-.
  11. Shaw LN, Jonsson I-M, Singh VK, Tarkowski A, Stewart GC. Inactivation of traP Has No Effect on the Agr Quorum-Sensing System or Virulence of Staphylococcus aureus. Infection and Immunity. 2007;75(9):4519-27.
  12. Kiran MD, Balaban N. TRAP Plays a Role in Stress Response in Staphylococcus Aureus. The International Journal of Artificial Organs. 2009;32(9):592-9.