The Lipoprotein Biogenesis Pathway: An Emerging Structural Target for Novel Antibiotics

A special group of proteins called Lipoproteins are essential to bacterial growth, disease and their resistance to antibiotic drugs. Lipoproteins require multiple stages of processing to allow their insertion, maturation and transportation to their final destination; equivalent to a bacterial factory-line.

2016 has proved to be an exciting year for studying this family of proteins. For the first time we have observed, in high definition, the molecular details of the first two check-point enzymes in the lipoprotein processing pathway, Lgt and LspA. On top of this, we have recently perceived two crucial structures of processed lipoproteins in their native complexes: namely BamABCDE and LptDE.

These complexes are of especial importance as they are responsible for the assembly of the fundamental components of the bacterial outer membrane, which constitutes the outermost fortifications. Meanwhile, the recently approved vaccines, Bexsero and Trumenba, for protection against meningitis B also contain lipoprotein components, NHBA and fHBP, illustrating the importance of these molecular structures.

To better understand these molecular machines, we ideally require well-defined structural information of these proteins, engaged with their native environment. However, this association incredibly difficult to capture due to the complexity of the membrane environment. Furthermore, the structures that are solved represent single-snapshot, static images with limited information about their normal motions.

We will use a range of computational biochemistry methods to study this pathway, while retaining our close engagement with structural biologists in readiness for further molecular structures and experimental data.

Our studies will permit the animation of the structures by incorporating their typical dynamic movements. In doing so, we will reveal how a protein embraces its native membrane environment in unprecedented detail. We can therefore understand how the substrates, proteins and known antibiotics interact in mechanistic detail.

By better understanding the precise details of this molecular pathway, we can strategically design novel drug inhibitors and therefore pave the way towards developing innovative antibiotics that will avert the antibiotic apocalypse.

Lipoproteins perform critical roles in bacterial physiology, pathogenicity, and antibiotic resistance. Their roles include modulation of the cell envelope structure, signal transduction and transport. Lipoproteins are processed by a pathway of membrane proteins - Sec, Lgt, LspA, Lnt and Lol - which insert, cleave and transport the protein substrate, while affixing lipid moieties to permit their tethering to the cell envelope. The recent determination of the three-dimensional protein structures of Lgt and LspA have enlivened lipoprotein research.

We will use a range of molecular simulation, bioinformatics and computational chemistry methods to study this pathway, while retaining our close engagement with structural biologists in readiness for further molecular structures and experimental data. Crucially our methods allow the incorporation of molecular dynamics, therefore the protein motions can be observed at the atomic level as the lipoprotein and processing machinery interact, while embracing their native membrane environment. Our studies also permit the accurate modelling of the lipid tethers at the N-terminal end of the protein and therefore reveal how these proteins are affixed to biological membranes. Furthermore, the bioinformatics methods permit the identification of both highly conserved residues, residue pairs and the modelling of unresolved components and mutants, while computational chemistry techniques permit the accurate docking of small molecules to the complexes and the prediction of reaction mechanisms for the intramembrane enzymes.

We expect that our data will greatly assist experimental scientists by permitting the design of knowledge-based assays and novel drug design strategies; revealing the suitability of this pathways as a target for novel antibiotics.

Who will benefit from this research?

This work is directly related to the BBSRC strategic priority areas of Bioscience for Health ("Develop and apply new tools in areas such as chemical biology, high resolution structural analysis"), to World-class Bioscience ("predictive, integrative and systems approaches in bioscience at a range of scales from molecules to...") and to Exploiting New Ways of Working by developing "the next generation of bioscience tools to drive new and deeper understanding in bioscience". This proposal directly relates to Combatting antimicrobial resistance by studying digital pathways within pathogenic organisms, while there are also elements to this proposal that comprise the systems approaches to and technology development for the biosciences. There is also a synthetic biology element, as the proteins of interest can also be manipulated for industrial biotechnology use. Therefore, this promotes partnerships with both pharmaceutical and biotechnology industries, in addition to the development of academic collaborations.

Thus, the beneficiaries will include:
1. The pharmaceutical industry and their stakeholders
2. The biotechnology sector and their stakeholders
3. Schools and Museums

How will they benefit from this research?

1. By inhibiting the proteins involved in lipoprotein biogenesis, maturation and localisation, one can develop novel antibiotics to control multi-drug-resistant strains of bacteria. Lipoproteins are also surface expressed in many bacteria and therefore these proteins are suitable candidates for novel vaccine design. Given, the drug discovery process can take up to 20 years, it is imperative we prepare well in advance to counteract the threat of a society where resistant bacterial strains are common-place and untreatable. Therefore, if we are sufficiently well equipped, the impact of this proposal will be long lasting and life-changing for the future generations. Our studies are also fundamental to better designing drugs that permeate more readily across biological membranes, thereby increasing their bioavailability. Our proposal will also enable more effective targeting of drugs to sites on membrane proteins 'buried' within the bilayer or otherwise an enhanced understanding of regions that are exposed to solvent and therefore more readily accessible. Both of these aspects are likely to increase the number of compounds that succeed in being developed, and thus have a significant commercial and a socio-economic impact.

2. The bio-nanotechnology sector are interested in lipoproteins, e.g. CsgG, and other membrane proteins as potential synthetic biosensors. An example of such a company is Oxford Nanopore Technologies. Improved understanding of the nature of protein/lipid interactions is likely to facilitate design of more stable membrane proteins for use in nanodevices.

3. Increased public understanding is an important benefit to the wider public. Computational approaches to the biosciences has a major advantage in that it is able to produce artistic and descriptive illustrations of biomolecules, that facilitate the accessibility of these ubiquitous macromolecular machines to the general public. Furthermore, molecular simulation enables the reanimation of statically resolved structures into movies that demonstrate the dynamics visually. By tuning the science to an appropriate level of detail, by using graphics tools such as Blender, one can make the research available as museum displays and as educational tools in schools.
Thus, the overall impact will be to advance UK knowledge and technological development as well as promoting health and wellbeing through better drug design. Ultimately this will add significantly to the competitiveness of UK industry.