Protein Structure and Folding
To be there when the picture is being painted
There is nothing quite like the excitement of discovery in science—of finding something no one else knew and seeing a story unfold. One has to be part of an emerging picture to feel the elation. These moments in a lifetime are few and far between, but they fuel enthusiasm and keep one going. They are embedded in struggles and joys of everyday life, years of establishing what Louis Pasteur called “the prepared mind,” working with mentors, trainees, and colleagues, failures and successes. This article recalls 1) how I got to be a biochemist; 2) my contributions as an educator and researcher, especially regarding meprin metalloproteases; and 3) my participation in communities of science.Exosites expedite blood coagulation
A careful balance between active-site and exosite contributions is critically important for the specificity of many proteases, but this balance is not yet defined for some of the serine proteases that serve as coagulation factors. Basavaraj and Krishnaswamy have closed an important gap in our knowledge of coagulation factor X activation by the intrinsic Xase complex by showing that exosite binding plays a critical role in this process, which they describe as a “dock and lock.” This finding not only significantly enhances our understanding of this step in the coagulation cascade and highlights parallels with the prothrombinase complex, but will also provide a novel rationale for inhibitor development in the future.PSI relieves the pressure of membrane fusion
Some plant proteases contain a latent sequence known as the plant-specific insert (PSI) that, upon release from the full protease sequence, initiates membrane fusion to defend from pathogens. However, the mechanism by which it exerts its effects has been unclear. Zhao et al. report an elegant integration of biophysical experiments and molecular dynamics simulations to reveal events leading up to PSI-mediated membrane fusion. Their results demonstrate a pH-dependent monomer-to-dimer transition, clear evidence of membrane association, and probable structures of prefusion intermediates.Securing SNAREs for assembly
SNARE proteins are essential for exocytosis, mediating the fusion of vesicles with their target membrane. Tethering factors play a key role in chaperoning SNARE assembly; however, the underlying molecular mechanisms are not well-understood. Here, Travis et al. report two crystal structures of a yeast tethering factor, the Dsl1 complex, bound with two SNARE proteins, revealing new insights into how tethering factors bridge vesicles to target membranes, recruit multiple SNARE proteins, trigger their conformational changes, and facilitate SNARE assembly.Forcing the ribosome to change its message
Mechanical forces can be generated when nascent protein segments are integrated into a membrane. These forces are then transmitted through the nascent protein to the ribosome's catalytic core, but only a few biological consequences of this process have been identified to date. In this issue, Harrington et al. present evidence that these forces form a conserved mechanism to influence the efficiency of ribosomal frameshifting during translation of viral RNA, indicating that mechanical forces may play a broader regulatory role in translation than previously appreciated.A checkpoint cliffhanger at the dawn of placental mammals
The PD-1 ligands PD-L1 and PD-L2 are commonly expressed on the surface of cells, where they regulate immune system activation. However, the specific role played by each ligand has been unclear. Using site-directed mutagenesis, surface plasmon resonance, and crystallography, Philips et al. explore the distinct features of PD-L2 and identify a specific evolutionary event linked to its appearance. This work provides a deeper understanding of how the immune system adapted to mammalian placental gestation and could be an important consideration in the development of new immune checkpoint therapies.Self-control of vitamin K2 production captured in the crystal
Menaquinone (MK) or vitamin K2 is an important metabolite that controls the redox/energy status of Mycobacterium tuberculosis. Although the major steps of MK biosynthesis have been delineated, the regulatory mechanisms of this pathway have not been adequately explored. Bashiri et al. now demonstrate that MenD, catalyzing the first committed step of MK production, is allosterically inhibited by a downstream cytosolic metabolite in the MK biosynthesis pathway.Integrated multidisciplinarity in the natural sciences
The integration of multiple perspectives in both the arts and natural sciences is tremendously powerful and arguably necessary for capturing relevant features of complex phenomena. Individual methods and models comprise abstractions from and idealizations of nature, and only the integration of multiple models, methods, and representations provides a means to reach more accurate results than relying on any single approach. In my Mildred Cohn Award Lecture at the 2019 ASBMB meeting, I illustrated the power of such multidisciplinary work by highlighting the successful integration of data and multiple views afforded by NMR spectroscopy, cryo-electron microscopy, cryo-electron tomography, X-ray crystallography, computation, and functional assays made possible through collaborative efforts by members of the Pittsburgh Center for HIV Protein Interactions.The long-awaited structure of HIPK2
Homeodomain-interacting protein kinases (HIPKs) are kinases that phosphorylate transcription factors involved in cell proliferation, differentiation, and apoptosis. Their structures have been long sought because of their potential as drug targets in cancers and fibrosis. Agnew and colleagues present the first crystal structure of the HIPK2 kinase domain, complexed with the small-molecule inhibitor CX-4945, revealing important structural differences from related protein kinases of the DYRK family.Liquid–liquid phase separation of tau protein: The crucial role of electrostatic interactions
Recent studies have indicated that tau, a protein involved in Alzheimer's disease and other neurodegenerative disorders, has a propensity to undergo liquid–liquid phase separation (LLPS). However, the mechanism of this process remains unknown. Here, we demonstrate that tau LLPS is largely driven by intermolecular electrostatic interactions between the negatively charged N-terminal and positively charged middle/C-terminal regions, whereas hydrophobic interactions play a surprisingly small role. Furthermore, our results reveal that, in contrast to previous suggestions, phosphorylation is not required for tau LLPS.Driving tau into phase-separated liquid droplets
Liquid–liquid phase separation of tau protein has been implicated in normal biological function as well as neurodegenerative diseases, including Alzheimer's. However, knowledge about these links is still scant, and the mechanisms driving tau into liquid droplets are poorly understood. A simplified in vitro system that uses unmodified human tau protein now suggests electrostatic interactions provide the basic instructions underlying liquid droplet formation.Isomerization as the secret Achilles' heel of long-lived proteins
Crystallin proteins, the dominant constituents of the eye lens, are prototypes of long-lived proteins. Such proteins can accumulate harmful modifications over their life span that render them prone to aggregation, which, in the case of lens crystallin, contributes to cataract formation. Lyon et al. now explore the structural and functional consequences of amino acid isomerization in α-crystallins using mass spectrometry, molecular dynamics simulations, and other strategies. Their results highlight the potential deleterious effects of these under-detected modifications on protein structural integrity and function.Human cytomegalovirus-specific T-cell receptor engineered for high affinity and soluble expression using mammalian cell display
T-cell receptors (TCR) have considerable potential as therapeutics and antibody-like reagents to monitor disease progression and vaccine efficacy. Whereas antibodies recognize only secreted and surface-bound proteins, TCRs recognize otherwise inaccessible disease-associated intracellular proteins when they are presented as processed peptides bound to major histocompatibility complexes (pMHC). TCRs have been primarily explored for cancer therapy applications but could also target infectious diseases such as cytomegalovirus (CMV).How a fortuitous collaboration helped catalyze new insights into helper proteins
What a way to begin a scientific career: Two newly independent researchers are brought together at a conference that was only possible because their country has just been reunified. In discussing their work, they discover that each holds half of a research project in their hands: The East German, Matthias Gaestel, can provide the critical protein for West German Johannes Buchner's robust assays. In their cross-country collaboration, they get clear and compelling data defining the previously enigmatic small heat shock proteins as molecular chaperones.Capturing Aβ42 aggregation in the cell
Novel imaging techniques with ever-increasing resolution are invaluable tools for the study of protein deposition, as they allow the self-assembly of proteins to be directly investigated in living cells. For the first time, the acceleration in Aβ42 aggregation induced by the Arctic mutation was monitored in cells, revealing a number of distinct morphologies that form sequentially. This approach will help discriminate the impacts of mutations on amyloid protein processing, Aβ aggregation propensity, and other mechanistic outcomes.An unexpected sticking point for carboxysome assembly
In order to improve photosynthetic efficiency, bacteria often enclose RubisCO and carbonic anhydrase into microcompartments called carboxysomes. Assembly of these complexes requires a protein called CcmM. It had previously been thought that CcmM mediated RubisCO assembly by displacing one of the RubisCO subunits, Ryan et al. show that despite having a three-dimensional structure that closely resembles the RubisCO small subunit, CcmM does not dislodge it, leading to a proposal for an alternative binding location.The Streptococcus gordonii Adhesin CshA Protein Binds Host Fibronectin via a Catch-Clamp Mechanism
Adherence of bacteria to biotic or abiotic surfaces is a prerequisite for host colonization and represents an important step in microbial pathogenicity. This attachment is facilitated by bacterial adhesins at the cell surface. Because of their size and often elaborate multidomain architectures, these polypeptides represent challenging targets for detailed structural and functional characterization. The multifunctional fibrillar adhesin CshA, which mediates binding to both host molecules and other microorganisms, is an important determinant of colonization by Streptococcus gordonii, an oral commensal and opportunistic pathogen of animals and humans.Barbiturates Bind in the GLIC Ion Channel Pore and Cause Inhibition by Stabilizing a Closed State
Barbiturates induce anesthesia by modulating the activity of anionic and cationic pentameric ligand-gated ion channels (pLGICs). Despite more than a century of use in clinical practice, the prototypic binding site for this class of drugs within pLGICs is yet to be described. In this study, we present the first X-ray structures of barbiturates bound to GLIC, a cationic prokaryotic pLGIC with excellent structural homology to other relevant channels sensitive to general anesthetics and, as shown here, to barbiturates, at clinically relevant concentrations.
