By Grace Langley
Proteins are prevalent in every second of our lives: they catalyse the chemical reactions in our bodies as enzymes; defend us against pathogens, as antibodies; and form the structure of our ears and noses, as collagen. It would be easy to assume we know everything there is to know about these complex molecules, coded indirectly by our DNA, but a recent study into the structure of a bacterial enzyme reveals a new chemical bond that influences protein structure and regulates protein function.
The Influence of Protein Structure
The structure of proteins is complex and multi-tiered, with each tier directly influencing the next. Their primary structure is coded for by the four bases of our DNA- adenosine, cytosine, thymine and guanine- in a process called protein synthesis. Each group of three bases - for example ACT or TTG- codes for a specific amino acid in the protein chain. Amino acids are biological molecules and there are only 20 different types that are naturally occurring in humans. Just like other biological molecules, we source amino acids from our diets and their main structures are identical save for an ‘R group’ which is the part of the amino acid that differentiates each amino acid from the next. Amino acids contain just a few core organic elements: carbon, hydrogen, oxygen and nitrogen, but the positioning and relationships between these elements -particularly in the R groups- is what creates the vast variety of protein structures that is vital to their specific functions. (Including the functions of enzymes, antibodies, structural proteins and hormones.) Peptide bonds are formed between the amino acids in the translation stage of protein synthesis, creating a long polymer chain, further bonds - including hydrogen bonding- form between the R groups of the amino acids, creating a complex and highly specific structure.
An infographic explanation of protein structure (Source: Made by the author- Grace Langley)
As shown in the graphic above, the overall structure of a protein is the result of a complex web of chemical bonds forming between amino acids that are not adjacent to each other in the polypeptide chain. These bonds are often referred to as crosslinks, and come in a few forms: ionic bonding between charged ions in the R groups; hydrogen bonds between hydrogen and elements such as nitrogen and oxygen; and disulfide bridges which are covalent bonds between two atoms of sulfur. But why is the structure of proteins so important to biological processes?
Let us focus on the example of enzymes for a moment. As biological catalysts, they increase the rate of every metabolic reaction, from aerobic respiration to the semi-conservative replication of DNA. This means enzymes are an integral part of the fundamental biochemistry of every organism, but in order to catalyse a reaction they must first interact with its reactants. In the case of enzymes, we call the reactants the substrates, and the interaction between an enzyme and a substrate is the formation of an enzyme-substrate complex. For an enzyme-substrate complex to form, the substrate physically has to fit into a section of the enzyme called the active site, so the active site of the enzyme and the substrate have to be a similar shape; in other words they have to be complementary. The shape of the active site of an enzyme is not simply random, it -like the rest of protein structure- is determined by the formation of crosslink bonds between amino acids and thus by the primary structure of the enzyme. Hence, the structure of an enzyme is vital to its functionality as a biological catalyst because it ensures that enzyme-substrate complexes can form.
However, not all chemical reactions are like aerobic respiration, which is constantly taking place in the mitochondria. There are some reactions that we only want to happen at certain times. One of these processes is the semiconservative replication of DNA, which is catalysed by the enzymes DNA helicase and DNA polymerase but only happens in the S phase of the cell cycle - a period in interphase where DNA is replicated. So how are enzymes regulated?
Enzyme Regulation and a New Crosslink Bond
The answer to enzyme regulation lies mostly in the disulfide bonding in proteins. Disulfides do not only help to maintain the 3D tertiary structure of proteins, but also sometimes regulate protein function. The formation of a disulfide bridge is a chemically reversible reaction, meaning that disulfide bridges can break and reform without requiring any additional resources. This unique property allows disulfide bridges to break and form freely. The breaking or formation of new disulfide bridges will change the structure of the enzyme, meaning the active site is no longer complementary to the substrate so the reaction can not be catalysed. This is the basic mechanism for protein regulation.
However, a recent publication in Nature (2.) by Wensien et al. reveals the recent discovery of a new type of crosslink that may also play a role in enzyme regulation. Wensien and her colleagues studied the enzyme trans aldolase in neisseria gonorrhoeae bacteria and observed the inactivity of the enzyme. Upon experimentation they discovered that if the enzyme was exposed to reducing agents (elements/ions that will donate electrons) that are normally used to break disulfide bridges, then the enzyme would become active. While this does not suggest anything beyond what was already believed about the role of disulfide bridges in enzyme regulation, the research team lead by Wensien then removed the amino acids that created the disulfide bridges, replacing them with other amino acids that would not form a disulfide crosslink.All the different mutants of transaldolase created, should have in theory, been active, and should not have been able to be inactivated. However, this was only the case for one of the mutants.
This unexpected behaviour, prompted the team to consider the possibility of disulfide bridges not being the only bonds present that help to regulate the activity of transaldolase, and in order to investigate further they turned to the technique of x-ray crystallography to examine the structure of the enzyme. X-ray crystallography can be conducted at a variety of different resolutions, but in this case was used at an atomic resolution, to analyse the individual elements in the structure of the transaldolase and their relationships with each other. The technique, which has led to the award of 28 Nobel prizes, revealed the new crosslink bond, believed to be responsible for the unexpected behaviour of the mutant enzymes.
The revealed crosslink is composed of nitrogen, oxygen and sulfur atoms, joined by covalent bonds in a similar way to the sulfur atoms in a disulfide bridge, and has been named by Wensien and her team, a N-O-S bridge.
The Chemistry of the N-O-S Bridge
We have already discussed the formation and breakage of a disulfide bridge as a reversible reaction, which previously scientists had attributed as the cause of its regulating ability. However, the N-O-S bridge seems to have a much more complex chemistry that will require further exploration by chemists and biologists alike. So what do we know already about the chemistry of the N-O-S bridge?
The covalent bond between nitrogen and oxygen theoretically requires very strong oxidising conditions, meaning an oxygen rich environment would be required for its formation, however these conditions would result in an oxidation of sulfur beyond what is present in the N-O-S bridge. What is immediately clear is that a N-O-S crosslink is formed in a different chemical reaction to how it is broken, as it is difficult to make but relatively easily broken.
The Future of the NOS Bridge
As part of their experimentation, Wensien and her team repeated x-ray crystallography on a variety of transaldolase enzymes from different organisms, and in doing so they discovered N-O-S bridges in the transaldolase enzyme of neisseria meningitidis and some human enzymes. Neisseria meningitidis is more commonly known as meningococcus and causes meningococcal meningitis and sometimes septicemia. There is speculation that the discovery by Wensien et al. may allow future development of drugs to target the N-O-S switch to inactivate the transaldolase enzyme of bacteria. Transaldolase is part of the pentose phosphate pathway (4.) and thus is a vital component in the synthesis of DNA. If a drug could be developed based on the N-O-S bridge to inhibit transaldolase enzymes, then DNA synthesis would be obstructed, theoretically preventing the division and multiplication of bacteria like meningococcus, and saving lives.
The future of the N-O-S switch is yet to be determined but, as is the way with science, there is sure to be a collective effort to discover more, and the research of Wensien and her team may shape the future of protein biochemistry, encouraging more teams to be adventurous with their use of x-ray crystallography and to consider the possibilities hidden within the complex structure of proteins.
Fass, D., & Semenov, S. N. (2021, May 5). Previously unknown type of protein crosslink discovered. Nature News. https://www.nature.com/articles/d41586-021-01135-3.
Wensien, M., Pappenheim, F. R. von, Funk, L.-M., Kloskowski, P., Curth, U., Diederichsen, U., … Tittmann, K. (2021, May 5). A lysine–cysteine redox switch with an NOS bridge regulates enzyme function. Nature News. https://www.nature.com/articles/s41586-021-03513-3.
Centers for Disease Control and Prevention. (2020, May 1). Meningococcal Disease (Neisseria meningitidis). Centers for Disease Control and Prevention. https://wwwnc.cdc.gov/travel/diseases/meningococcal-disease.
GA;, S. A. K. S. (2009). Transaldolase: from biochemistry to human disease. The international journal of biochemistry & cell biology. https://pubmed.ncbi.nlm.nih.gov/19401148/.