Protein structure, dynamics, and function—a 20th IUPAB Congress symposium

Abstract

A wide range of topics was raised by the four invited speakers who took part in the session on protein structure, dynamics, and function during the 20th IUPAB Congress. Most of the emphasis was placed on understanding the underlying biological phenomena of interest although applications in drug development were also mentioned. For both these purposes, it was clear that a complete description of the dynamics of the system was as important as the structures themselves. The subjects covered included antibiotic peptides, sodium channels, the synthesis of the bacterial cell wall, and protein dynamics using X-FELs.

The session on protein structure, dynamics, and function, which took place during the 20th Congress of the International Union of Pure and Applied Biophysics, was lively and well attended and included plenty of interaction from the on-line attendees. The very broad nature of the subject matter meant that it overlapped significantly with several of the other sessions. Among others, the sessions on Ionic channels and membrane transporters, on macromolecular machines and switching devices, and on biomolecular association and dynamics come readily to mind along with the symposium on protein folding, misfolding, and unfolding. Furthermore, since the association between membranes and proteins/peptides was also a recurrent theme, the session also integrated well with other parts of the congress. Molecular recognition was the central focus and how an increase in the biophysical techniques available for probing systems at the molecular level is rapidly increasing our understanding of complex biological phenomena.

The session was well balanced in terms of gender and geography. We were lucky to be able to count on one speaker from Australia, one from the USA, one from Europe, and one who holds joint appointments in both Europe and South America. All speakers are active research scientists who work outside their countries of origin and there was, therefore, a “border-free” feeling to the event. Unsurprisingly, the relevance of structural information for the development of novel drugs was a recurrent theme throughout the session. However, there was as much (or more) emphasis on the importance of understanding the basic underlying biology as there was on the bioactive compounds themselves. This gave the session an invigorating gust of fresh air in support of basic science.

Frances Separovic got the session off to a good start exemplifying many of these points. Her studies of bioactive peptides, and particularly Maculatin 1.1 from Australian tree frogs, were inspired by the rising tide of antibacterial resistance and the need for new antibiotics. Antimicrobial peptides are seen as a potential alternative to small molecules and for their development there is a need for a better understanding of the peptide-lipid interactions involved (Sani and Separovic 2016). Dr. Separovic elegantly dissected these interactions in the case of Maculatin 1.1 by employing a series of biophysical techniques, principally NMR and CD, together with molecular dynamics simulations (Separovic et al. 2020). She initially honed in on the important role played by anionic lipids which are predominant in Gram positive bacteria, against which Maculatin 1.1 is most effective. She also discussed how the length of the lipids can influence the tipping point between different modes of interaction. She went on to outline experiments designed to use the full potential of NMR together with MD simulations to establish the helical structure of the peptide within the membrane and its oligomeric arrangement which leads to pore formation (Sani et al. 2019). In the final part of her presentation, she mentioned some proof of concept experiments in vivo which appear to represent a new direction for future research (Overall et al. 2019; Separovic et al. 2020).

Marius Schmidt changed gear somewhat and confronted the difficulties of studying protein dynamics directly—as he nicely puts it “molecular movies” of proteins in action. He initially described the unique properties which an X-FEL provides for such studies, related to the intensity and time structure of the radiation which allow for the use of showers of very small crystals (< 1000 times the volume of those typically used with conventional sources). He moved on to describe several examples of the application of this approach, initially focusing on light-sensitive proteins. In the case of the photoactive yellow protein, where direct observation of the all-important trans–cis isomerization was missing, he described the use of pump-probe experiments to obtain very clear difference maps (Pande et al. 2016). With these, he was able to explain the structural transition itself as well as determining the timescale over which it occurs. In a second example, the role of a conformational change within the chloride channel was elegantly used to explain the pumping mechanism across the membrane (Yun et al. 2021). However, most biological systems are not light-sensitive, so a rapid mix and inject strategy needs to be employed (Pandey et al. 2021). The elegant movies which result readily allow for visualizing changes occurring to both substrate and enzyme during catalysis. Schmidt subsequently applied the pump probe approach to a phytochrome and made it clear that the interpretation of the resulting maps in terms of the chemistry involved is not always trivial (Carrillo et al. 2021). Finally, he touched on the possibility of eliminating the crystal altogether and using X-FELs for single particle experiments. Throughout his talk, it became clear that there is still a need for constant improvements to the instrumentation (both the source itself and the auxiliary equipment) in order to extract as much biological information as possible. Further details can be found in his review paper which is part of this special issue.

Bonnie Wallace brought us back to membrane proteins and specifically the voltage gated sodium channels, once again with a focus on their potential as drug targets. With beautiful clarity, Wallace provided an overview of the structures of both mammalian and bacterial sodium channels. Because of their relevance as drug targets, much of her talk was devoted to the identification of potentially druggable pockets. A hydrophobic fenestration at the center of the channel has been described in the past but Wallace proposed to look beyond this for other alternatives. By using a combination of the information coming from the structures themselves together with prior knowledge of drugs which bind to the sodium channel (such as antiepilectics, analgesics, or even off-target examples such as tamoxifen), several novel opportunities emerged (Sula et al. 2021; Zanatta et al. 2019). Subsequent studies in which the structures of complexes formed between the channel and the chosen drugs revealed the diversity of binding sites available, thus fulfilling Wallace’s speculation that many possibilities may lie beyond the fenestrations identified many years earlier.

The focus of Andrea Dessen’s talk was on the peptidoglycan of the bacterial cell wall and particularly its biosynthesis. Penicillin-binding proteins (PBPs) are fundamental for this process as well as being the target for penicillin-like antibiotics. Once again, the motivation behind much of Dessen’s work is to apply the results of her basic research in the development of novel therapies. By using a Y2H approach, a protein called MreC, a component of the elongasome, was identified as a PBP2 binding partner important for the production of the cell wall in non-spherical bacteria, probably serving as a scaffold. Dessen used both cryo-EM and traditional X-ray diffraction to reveal the way in which MreC forms polymeric scaffolds with which PBP2 specifically interacts, inducing a large movement in the latter (Contreras-Martel et al. 2017). Studies with mutants allowed her to dissect the MreC structure and elaborate a model in which cell wall synthesis is controlled by different modes of MreC self-association (Martins et al. 2021). This can be either in the form of low molecular weight dimers or as polymeric tubes based on six pairs of antiparallel filaments. Dessen finalized with a warning concerning the threat posed by resistant strains of the bacterium Pseudomonas aeruginosa, which appears to be able to change shape in the presence of β-lactam antibiotics as a consequence of dissolving its cell wall as a survival mechanism. Her work on the fundamental structural biology of the synthesis of the cell wall therefore aids in establishing the foundations for further studies, particularly those directed towards novel anti-biotic development.

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