Crystal structure of the hemoglobin dodecamer from Lumbricus erythrocruorin: allosteric core of giant annelid respiratory complexes

Erythrocruorins are highly cooperative giant extracellular respiratory complexes found in annelids, where they serve the same function as red blood cells. Our previous 5.5A resolution crystal structure of Lumbricus terrestris erythrocruorin revealed a hierarchical organization of 144 oxygen-binding hemoglobin chains that are assembled into 12 dodecamers arranged at the periphery of the complex around a central scaffold formed by 36 non-hemoglobin subunits. We present here the 2.6A resolution crystal structure of the Lumbricus hemoglobin dodecameric subassembly, which provides the first atomic models of the erythrocruorin allosteric core. The hemoglobin dodecamer has a molecular 3-fold axis of symmetry that relates three heterotetramers, each of which is composed of two tightly associated heterodimers. The structure reveals details of the interfaces, including key side-chain interactions likely to contribute to ligand-linked allosteric transitions, and shows the crowded nature of the ligand-binding pockets. Comparison of the Lumbricus dimeric assemblies with similar ones from mollusks and echinoderms suggests plausible pH-dependent quaternary transitions that may occur in response to proton binding and ligand release. Thus, these results provide the first step towards elucidating the structural basis for the strong allosteric properties of Lumbricus erythrocruorin

Synthesis of Aspartame by Thermolysin: An X‑ray Structural Study

Protease mediated peptide synthesis (PMPS) was first described in the 1930s but remains underexploited today. In most PMPS, the reaction equilibrium is shifted toward synthesis by the aqueous insolubility of product generated. Substrates and proteases are selected by trial and error, yields are modest, and reaction times are slow. Once implemented, however, PMPS reactions can be simple, environmentally benign, and readily scalable to a commercial level. We examined the PMPS of a precursor of the artificial sweetener aspartame, a multiton peptide synthesis catalyzed by the enzyme thermolysin. X-ray structures of thermolysin in complex with aspartame substrates separately, and after PMPS in a crystal, rationalize the reaction’s substrate preferences and reveal an unexpected form of substrate inhibition that explains its sluggishness. Structure guided optimization of this and other PMPS reactions could expand the economic viability of commercial peptides beyond current high-potency, low-volume therapeutics, with substantial green chemistry advantages

Lumbricus erythrocruorin at 3.5 A resolution: architecture of a megadalton respiratory complex

Annelid erythrocruorins are highly cooperative extracellular respiratory proteins with molecular masses on the order of 3.6 million Daltons. We report here the 3.5 A crystal structure of erythrocruorin from the earthworm Lumbricus terrestris. This structure reveals details of symmetrical and quasi-symmetrical interactions that dictate the self-limited assembly of 144 hemoglobin and 36 linker subunits. The linker subunits assemble into a core complex with D(6) symmetry onto which 12 hemoglobin dodecamers bind to form the entire complex. Although the three unique linker subunits share structural similarity, their interactions with each other and the hemoglobin subunits display striking diversity. The observed diversity includes design features that have been incorporated into the linker subunits and may be critical for efficient assembly of large quantities of this complex respiratory protein

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Third International Conference on 3Rs Research and Progress, Vishakhapatnam, 2022.

Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Al- ternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in- person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day