Ian B. Robertson

Cytochrome c assembly: A tale of ever increasing variation and mystery?

Formation of cytochromes c requires a deceptively simple post-translational modification, the formation of two thioether bonds (or rarely one) between the thiol groups of two cysteine residues found in a CXXCH motif (with some occasional variations) and the vinyl groups of heme. There are three partially characterised systems for facilitating this post-translational modification; within these systems there is also variation. In addition, there are clear indications for two other distinct systems. Here some of the current issues in understanding the systems are analysed.

Structure of the Fibrillin-1 N-Terminal Domains Suggests that Heparan Sulfate Regulates the Early Stages of Microfibril Assembly

Summary The human extracellular matrix glycoprotein fibrillin-1 is the primary component of the 10- to 12-nm-diameter microfibrils, which perform key structural and regulatory roles in connective tissues. Relatively little is known about the molecular mechanisms of fibrillin assembly into microfibrils. Studies using recombinant fibrillin fragments indicate that an interaction between the N- and C-terminal regions drives head-to-tail assembly. Here, we present the structure of a fibrillin N-terminal fragment comprising the fibrillin unique N-terminal (FUN) and the first three epidermal growth factor (EGF)-like domains (FUN-EGF3). Two rod-like domain pairs are separated by a short, flexible linker between the EGF1 and EGF2 domains. We also show that the binding site for the C-terminal region spans multiple domains and overlaps with a heparin interaction site. These data suggest that heparan sulfate may sequester fibrillin at the cell surface via FUN-EGF3 prior to aggregation of the C terminus, thereby regulating microfibril assembly.

Dispensable residues in the active site of the cytochrome c biogenesis protein CcmH.

CcmH functions in the assembly of c‐type cytochromes in the Escherichia coli periplasm. The conserved cysteine pair in the N‐terminal of its two membrane‐anchored periplasmic domains is thought to reduce the CXXCH motif of cytochromes c. The recent structure of Pseudomonas aeruginosa CcmH identified conserved residues that might be functionally important. We replaced with alanine the active‐site cysteines of E. coli CcmH, as well as R42, S54, R63, and tested the effects on cytochrome c production anaerobically and aerobically. Unexpectedly, replacement of the conserved non‐cysteine active‐site residues had little effect, whilst the cysteines were required under aerobic, but not anaerobic, conditions. We confirmed that removal of the C‐terminal tetratricopeptide‐like domain does not, surprisingly, abolish assembly of cytochromes c.

NMR Spectroscopic and Bioinformatic Analyses of the LTBP1 C-Terminus Reveal a Highly Dynamic Domain Organisation

Proteins from the LTBP/fibrillin family perform key structural and functional roles in connective tissues. LTBP1 forms the large latent complex with TGFβ and its propeptide LAP, and sequesters the latent growth factor to the extracellular matrix. Bioinformatics studies suggest the main structural features of the LTBP1 C-terminus are conserved through evolution. NMR studies were carried out on three overlapping C-terminal fragments of LTBP1, comprising four domains with characterised homologues, cbEGF14, TB3, EGF3 and cbEGF15, and three regions with no homology to known structures. The NMR data reveal that the four domains adopt canonical folds, but largely lack the interdomain interactions observed with homologous fibrillin domains; the exception is the EGF3-cbEGF15 domain pair which has a well-defined interdomain interface. 15N relaxation studies further demonstrate that the three interdomain regions act as flexible linkers, allowing a wide range of motion between the well-structured domains. This work is consistent with the LTBP1 C-terminus adopting a flexible “knotted rope” structure, which may facilitate cell matrix interactions, and the accessibility to proteases or other factors that could contribute to TGFβ activation.

Evolutionary Insights into Fibrillin Structure and Function in the Extracellular Matrix

The 10–12 nm diameter microfibrils of the extracellular matrix are vital components of dynamic tissues, playing a major role in providing the tissues of metazoan species with their biomechanical properties. They also have functional roles in the development of elastic fibres, as a scaffold for the deposition of tropoelastin, and in the regulation of growth factors. Fibrillins are large, ~350 kDa glycoproteins that assemble to form the microfibrils. Throughout evolution, the fibrillins show a remarkably conserved domain organisation, dominated by calcium-binding epidermal growth factor domains interspersed with transforming growth factor-β-binding protein-like domains. Mutations in the fibrillin genes cause a range of human diseases including Marfan syndrome, Beals syndrome, stiff skin syndrome and the acromelic dysplasias, which affect the skin, skeleton, ocular and cardiovascular systems. In recent years, studies using molecular, cellular and animal models have yielded new information on the processes of microfibril assembly and organisation and how their regulation of growth factors is involved in disease pathogenesis. Recent advances in bioinformatics and the availability of whole genome sequences are now providing new insights into the functions of different regions of the fibrillin polypeptides, helping us to understand how fibrillin structure and function has evolved as the extracellular matrix has increased in complexity.

Responsible Investment Requires a Proxy Voting System Responsive to Retail Investors

There is growing awareness amongst retail investors of the importance of environmental, social, and governance (ESG) factors to the performance of their stocks. The same factors impact their lives from a broader societal and economic perspective. Institutional investors have incorporated ESG issues into their proxy voting and corporate engagement. Retail investors who invest in stocks directly have the same voting rights, and collectively a similar power, but data shows that their voting rates have declined precipitously over the past forty years. This chapter traces the history of property rights and proxy voting, examines them within the current regulatory context, and posits that economic rights have been well protected but ownership rights have been neglected. An established framework for stages of capitalism is re-imagined, situating retail investors’ disengagement from the proxy process and highlighting suggestions to regulators for addressing the proxy voting gap.

The N-Terminal Region of Fibrillin-1 Mediates a Bipartite Interaction with LTBP1

Summary Fibrillin-1 (FBN1) mutations associated with Marfan syndrome lead to an increase in transforming growth factor β (TGF-β) activation in connective tissues resulting in pathogenic changes including aortic dilatation and dissection. Since FBN1 binds latent TGF-β binding proteins (LTBPs), the major reservoir of TGF-β in the extracellular matrix (ECM), we investigated the structural basis for the FBN1/LTBP1 interaction. We present the structure of a four-domain FBN1 fragment, EGF2-EGF3-Hyb1-cbEGF1 (FBN1E2cbEGF1), which reveals a near-linear domain organization. Binding studies demonstrate a bipartite interaction between a C-terminal LTBP1 fragment and FBN1E2cbEGF1, which lies adjacent to the latency-associated propeptide (LAP)/TGF-β binding site of LTBP1. Modeling of the binding interface suggests that, rather than interacting along the longitudinal axis, LTBP1 anchors itself to FBN1 using two independent epitopes. As part of this mechanism, a flexible pivot adjacent to the FBN1/LTBP1 binding site allows LTBP1 to make contacts with different ECM networks while presumably facilitating a force-induced/traction-based TGF-β activation mechanism.