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Restricted accessIntroductionFirst published December, 2019pp. 1507-1509
, Rhett A Kovall
Restricted accessReview articleFirst published December, 2019pp. 1510-1519ADAM (a disintegrin and metalloproteinase) proteins are type-1 transmembrane and secreted proteins that function in cell adhesion and signal transduction. Here we review the structural features of ADAM proteins that direct their biological functions in ectodomain shedding and cell adhesion.
Recent structural advances have provided a deeper appreciation for interdomain relationships that modulate the activity of ADAM proteins in ectodomain shedding and cellular adhesion. Our review covers these new findings, and places them into historical context. The new results make clear that the metalloproteinase domain works in combination with its ancillary domains to execute its biological function. The ADAM ectodomain is dynamic, and accesses conformations that require interdomain movements during its enzymatic “lifecycle.” Fundamental questions about ADAM activation and substrate selection, however, still remain unanswered. Elucidating the biochemical and structural basis for ADAM regulation will be an exciting avenue of future research that should greatly advance our understanding of ADAM function in biology and human pathogenesis.
Restricted accessReview articleFirst published December, 2019pp. 1520-1529, Rhett A KovallThis mini review discusses the protein complexes comprised of the universal Notch signaling transcription factor, CSL (CBF1/Su(H)/Lag-1), and its activating or repressing transcriptional coregulation partners. Many of these complex structures have been solved crystallographically as well as undergoing extensive binding studies with wild-type and mutant variants. Notch signaling is critically important in a large variety of basic biological processes: cell proliferation, differentiation, cell cycle control to name a few. Aberrant Notch thus remains a coveted target for pharmaceutical intervention. To that end, we provide a molecular-level summary of the similarities and differences in the Notch coregulator complexes that ultimately govern these processes. We highlight a conserved binding motif that multiple superficially unrelated proteins have adopted to become involved in Notch target gene regulation. As CSL-interacting small molecules begin to be characterized, this review will provide insight to potential binding sites and differential complex disruption.
Proper Notch signaling regulation is informed by many distinct protein complexes involving a single nuclear effector. A decade of research into these protein complexes yields multiple crystal structures and a wealth of binding data to guide drug development for Notch-related diseases – cancer, cardiovascular, development disorders.
Restricted accessReview articleFirst published December, 2019pp. 1530-1546The transforming growth factor beta (TGFβ) signaling pathway orchestrates a wide breadth of biological processes, ranging from bone development to reproduction. Given this, there has been a surge of interest from the drug development industry to modulate the pathway – at several points. This review discusses and provides additional context for several layers of the TGFβ signaling pathway from a structural biology viewpoint. The combination of structural techniques coupled with biophysical studies has provided a foundational knowledge of the molecular mechanisms governing this high impact, ubiquitous pathway, underlying many of the current therapeutic pursuits. This work seeks to consolidate TGFβ-related structural knowledge and educate other researchers of the apparent gaps that still prove elusive. We aim to highlight the importance of these structures and provide the contextual information to understand the contribution to the field, with the hope of advancing the discussion and exploration of the TGFβ signaling pathway.
The transforming growth factor beta (TGFβ) signaling pathway is a multifacetted and highly regulated pathway, forming the underpinnings of a large range of biological processes. Here, we review and consolidate the key steps in TGFβ signaling using literature rooted in structural and biophysical techniques, with a focus on molecular mechanisms and gaps in knowledge. From extracellular regulation to ligand–receptor interactions and intracellular activation cascades, we hope to provide an introductory base for understanding the TGFβ pathway as a whole.
Restricted accessReview articleFirst published December, 2019pp. 1547-1558Betaglycan and endoglin, membrane-bound co-receptors of the TGF-β family, are required to mediate the signaling of a select subset of TGF-β family ligands, TGF-β2 and InhA, and BMP-9 and BMP-10, respectively. Previous biochemical and biophysical methods suggested alternative modes of ligand binding might be responsible for these co-receptors to selectively recognize and potentiate the functions of their ligands, yet the molecular details were lacking. Recent progress determining structures of betaglycan and endoglin, both alone and as bound to their cognate ligands, is presented herein. The structures reveal relatively minor, but very significant structural differences that lead to entirely different modes of ligand binding. The different modes of binding nonetheless share certain commonalities, such as multivalency, which imparts the co-receptors with very high affinity for their cognate ligands, but at the same time provides a mechanism for release by stepwise binding of the signaling receptors, both of which are essential for their functions.
The TGF-β family is one of the most highly diversified signaling families, with essential roles in nearly all aspects of metazoan biology. Though functionally diverse, all 33 human TGF-β family ligands signal through a much more limited number of receptors. Thus the signaling repertoire is limited and cannot account for the functional diversity of signaling ligands
Restricted accessBrief reportFirst published December, 2019pp. 1559-1567The growing availability of complex structures in the Protein Data Bank has provided key insight into the molecular architecture of protein–protein interfaces. The remarkable diversity observed in protein binding modes is paralleled by a tremendous variation in binding affinities, with interaction half-lives ranging from days to milliseconds. Within the protein interactome, low-affinity binding events have been particularly difficult to visualize by traditional structural methods, which has spurred the development of innovative strategies for reconstituting these short-lived yet biologically essential assemblies. An important takeaway from structural studies of low-affinity systems is that there is no universal solution for stabilizing protein complexes, and approaches such as single-chain fusions, biochemical linkages, and affinity-maturation have each been successful in certain contexts. In this article, we review how advances in molecular engineering have been used to capture weakly associated complexes for structure determination, and we provide perspectives on how the continued application of these methods can shed new light on the “hidden world” of low-affinity interactions.
Low-affinity protein interactions, while biologically essential, have been difficult to visualize by traditional methods in structural biology. In this review, we describe a series of innovative molecular engineering strategies that have been used to stabilize weakly bound protein complexes for structure determination. By highlighting several examples from the literature along with potential advantages and disadvantages of the individual approaches, we hope to provide an introductory resource for structural biologists studying low-affinity systems.
Restricted accessReview articleFirst published December, 2019pp. 1568-1576Since their discovery just over 25 years ago, the single variable domain from heavy-chain-only antibodies plays a role in an increasing number of antibody-based applications. Structural and biophysical studies have revealed that the small, ∼15 kDa, single variable domain found in camelids displays versatility in target recognition. Such insight has served as the foundation to develop and engineer VHH domains with enhanced properties capable of targeting a range of therapeutically relevant protein antigens or low-molecular weight haptens. Furthermore, the modular nature of VHH domains allows them to be introduced into constructs that are simply not possible with conventional antibodies. Here, we review the structural and biophysical properties of VHH domains, highlight recent VHH-based therapeutics and diagnostics, and provide insight into VHH engineering that may pave the way to next-generation single domain antibody applications.
The development of novel antibody formats, beyond conventional antibodies, opens new possibilities in medical therapies, diagnostics, and general life science applications requiring affinity reagents. The camelid VHH domain, from heavy-chain-only antibodies, has emerged as a jack-of-all-trades module for novel affinity reagents. Applications include targeted cancer therapies, novel antimicrobial agents, conformation specific reagents, and tailor-made molecular switch entities. The breadth of unique uses for the VHH will continue to grow, opening new opportunities to treat and understand disease.
Restricted accessReview articleFirst published December, 2019pp. 1577-1583With the emergence of immuno-oncology, new therapeutic agents that modulate immune activation and regulation are being used to treat cancer patients with durable response. It is well known that following T-cell receptor (TCR) activation, many co-receptors can augment or suppress the TCR signal, and therapeutically targeting these co-receptors has proven effective. The B7-CD28 family is comprised of such immune-regulatory receptors, and antibodies against its members programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have revolutionized cancer treatment. These therapies promote an immune response against tumor cells, which demonstrated better long-term survival and tolerability compared to traditional cancer treatments. In this review we describe the history of the expanding B7-CD28 family, and by comparison of sequence and structure reveal that it is a non-traditional family. The family has grown to include proteins that share low sequence identity, generally grouped by regulation of immune response, which utilize the common immunoglobulin fold. This low level of commonality has provided additional challenges to the drug discovery process as the mechanisms and therapeutic potency between family members can vary greatly.
Immunotherapy as a field has dramatically expanded in the last decade in the area of oncology with efficacy demonstrated by PD-1, PD-L1, and CTLA-4 blockade. With all three “checkpoint blockade” receptors being in the B7-CD28 family, there has been increased interest in targeting other members in this family due to redundancy in immune regulation, i.e., the combination of therapeutic agents targeting multiple co-inhibitory receptors may yield additional antitumor efficacy. Therefore significant resources are being dedicated to developing additional B7-CD28 treatment options.
Open accessReview articleFirst published December, 2019pp. 1584-1595Huntington’s disease, like other neurodegenerative diseases, continues to lack an effective cure. Current treatments that address early symptoms ultimately fail Huntington’s disease patients and their families, with the disease typically being fatal within 10–15 years from onset. Huntington’s disease is an inherited disorder with motor and mental impairment, and is associated with the genetic expansion of a CAG codon repeat encoding a polyglutamine-segment-containing protein called huntingtin. These Huntington’s disease mutations cause misfolding and aggregation of fragments of the mutant huntingtin protein, thereby likely contributing to disease toxicity through a combination of gain-of-toxic-function for the misfolded aggregates and a loss of function from sequestration of huntingtin and other proteins. As with other amyloid diseases, the mutant protein forms non-native fibrillar structures, which in Huntington’s disease are found within patients’ neurons. The intracellular deposits are associated with dysregulation of vital processes, and inter-neuronal transport of aggregates may contribute to disease progression. However, a molecular understanding of these aggregates and their detrimental effects has been frustrated by insufficient structural data on the misfolded protein state. In this review, we examine recent developments in the structural biology of polyglutamine-expanded huntingtin fragments, and especially the contributions enabled by advances in solid-state nuclear magnetic resonance spectroscopy. We summarize and discuss our current structural understanding of the huntingtin deposits and how this information furthers our understanding of the misfolding mechanism and disease toxicity mechanisms.
Many incurable neurodegenerative disorders are associated with, and potentially caused by, the amyloidogenic misfolding and aggregation of proteins. Usually, complex genetic and behavioral factors dictate disease risk and age of onset. Due to its principally mono-genic origin, which strongly predicts the age-of-onset by the extent of CAG repeat expansion, Huntington’s disease (HD) presents a unique opportunity to dissect the underlying disease-causing processes in molecular detail. Yet, until recently, the mutant huntingtin protein with its expanded polyglutamine domain has resisted structural study at the atomic level. We present here a review of recent developments in HD structural biology, facilitated by breakthrough data from solid-state NMR spectroscopy, electron microscopy, and complementary methods. The misfolded structures of the fibrillar proteins inform our mechanistic understanding of the disease-causing molecular processes in HD, other CAG repeat expansion disorders, and, more generally, protein deposition disease.
Restricted accessReview articleFirst published December, 2019pp. 1596-1604The hydrolysis of last resort carbapenem antibiotics by
The work herein is important to the field as it provides a clear and succinct accounting of available KPC-2 structures. The work advances the field by collecting and analyzing differences and similarities across the available structures. This work features new analyses and interpretations of the existing structures which will impact the field in a positive way by making structural insights more widely available among the beta-lactamase community.