Biology subjects

Michie, K. A.

Publications and source records attributed to Michie, K. A..

4 recordsLinked to original sources

bioRxiv2024

Assessing the Validity of Leucine Zipper Constructs Predicted in AlphaFold2.

AP-1 transcription factors are a network of cellular regulators, that combine in different dimer pairs to control a range of pathways involved in differentiation, growth, and cell death. They dimerise via leucine zipper coiled-coil domains, that are preceded by a basic DNA binding domain. Depending on which AP-1 transcription factors dimerise, different DNA sequences will be recognised resulting in differential gene expression. The affinity of AP-1 transcription factors for each other dictates which dimers form. The relative concentration of AP-1 transcription factors varies with tissue type and environment, adding another layer of control to this integral network of cellular regulation. The development of artificial intelligence (AI) protein structure prediction programs gives us a new technique to investigate or predict how dimerization effects combinatorial control. AlphaFold2 and AlphaFold-Multimer are AI programs that predict 3D structures of proteins using primary sequence as their only input, even if there is no homologous model available. To fully realise the potential of AI for structural biology, it is essential to understand its current capabilities and limitations. In this study we used the classical example of an AP-1 dimer: Fos and Jun, to interrogate how AlphaFold2 and AlphaFold-Multimer model leucine zipper domains, and if AlphaFold-Multimer can be used to differentiate between probable and improbable dimer interfaces. We found that AlphaFold-Multimer predicts highly confident leucine zipper dimers, even for dimer pairs, such as the FosB homodimer, for which electrostatics are known to prevent their formation in vivo. This is an important case study concerning high-confidence, but low-accuracy protein structure prediction. statementArtificial intelligence (AI) programs that predict protein structures, like AlphaFold, could transform structural biology by speeding up the experimental process. However, it is important to grasp the capabilities and limitations of these AI tools. This study examines how AlphaFold identifies structural features, specifically a leucine zipper, while not considering other factors like electrostatic interactions, using the well-studied transcription factors Fos and Jun as a case study.

bioinformatics↗

bioRxiv2024

Cell division protein A (CdpA) assembles and anchors the two FtsZ rings at midcell in haloarchaea

Many archaea appear to divide through the coordinated activities of two FtsZ homologues (FtsZ1 and FtsZ2) and another bacterial cell division homologue (SepF), which are part of the midcell division ring. Here, we identify an additional protein (HVO_0739, renamed CdpA) that is involved in cell division in Haloferax volcanii, with homologues in other Haloarchaea. CdpA localizes at the mid-cell division ring, and this requires the presence of the ring-assembly protein FtsZ1. The division constriction protein FtsZ2 also influenced the proper midcell assembly and structure of CdpA. In the absence of CdpA, cells frequently failed to divide properly, and FtsZ1 formed poorly condensed pseudo-helical structures spanning across a broad region of the cell, whereas FtsZ2 showed mispositioned foci, nano-rings, and filaments. The rate of directional movement of FtsZ1 and FtsZ2 structures around the division ring appeared minimally affected by loss of CdpA, which resulted in continual repositioning of the aberrant FtsZ structures in the cells. In contrast to the FtsZ proteins, CdpA formed relatively immobile foci around the ring. Protein domain function studies, pull-down assays, and multimer structure predictions suggest that CdpA is part of a membrane complex that tethers FtsZ2 and other division proteins to the midcell membrane. Our discovery of an archaeal FtsZ organisation and midcell anchor protein offers new insights into cell division mechanisms that are similar across the tree of life.

microbiology↗

bioRxiv2023

Molecular dissection of the soluble photosynthetic antenna from a cryptophyte alga

Cryptophyte algae have a unique phycobiliprotein light-harvesting antenna that fills a spectral gap in chlorophyll absorption, however, it is unclear how it transfers energy efficiently to photosystems. We show that the cryptophyte Hemiselmis andersenii expresses an energetically complex antenna comprising three distinct spectrotypes of phycobiliprotein with different quaternary structures arising from a diverse subunit family. The bulk of the antenna consists of open quaternary form phycobiliproteins acting as primary photon acceptors, supplemented by novel open-braced forms. The final components are closed forms with a long wavelength spectral feature due to substitution of a single chromophore. We propose that the macromolecular organization of the cryptophyte antennas consists of bulk open and open-braced forms that transfer excitations to photosystems via this bridging closed form phycobiliprotein. One-Sentence SummaryAlgae generate a rainbow of antenna proteins by combining a conserved subunit with different members of a multigene family.

molecular biology↗

bioRxiv2022

Ancestral reconstruction of the MotA stator subunit reveals that conserved residues far from the pore are required to drive flagellar motility

The bacterial flagellar motor (BFM) is a rotary nanomachine powered by the translocation of ions across the inner membrane through the stator complex. The stator complex consists of two membrane proteins: MotA and MotB (in H+ powered motors), or PomA and PomB (in Na+ powered motors). In this study we used ancestral sequence reconstruction (ASR) to probe which residues of MotA correlate with function and may have been conserved to preserve motor function. We reconstructed ten ancestral sequences of MotA and found four of them were motile in combination with contemporary E. coli MotB and in combination with our previously published functional ancestral MotBs. Sequence comparison between wild-type (WT) E. coli MotA and MotA-ASRs revealed 30 critical residues across multiple domains of MotA that were conserved among all motile stator units. These conserved residues included pore-facing, cytoplasm-facing and MotA-MotA intermolecular facing sites. Overall, this work demonstrates the role of ASR in assessing conserved variable residues in a subunit of a molecular complex.

microbiology↗