Co-Evolution of Opioid and Adrenergic Ligands and Receptors: Shared, Complementary Modules Explain Evolution of Functional Interactions and Suggest Novel Engineering Possibilities

Cross-talk between opioid and adrenergic receptors is well-characterized and involves second messenger systems, the formation of receptor heterodimers, and the presence of extracellular allosteric binding regions for the complementary ligand; however, the evolutionary origins of these interactions have not been investigated. We propose that opioid and adrenergic ligands and receptors co-evolved from a common set of modular precursors so that they share binding functions. We demonstrate the plausibility of this hypothesis through a review of experimental evidence for molecularly complementary modules and report unexpected homologies between the two receptor types. Briefly, opioids form homodimers also bind adrenergic compounds; opioids bind to conserved extracellular regions of adrenergic receptors while adrenergic compounds bind to conserved extracellular regions of opioid receptors; opioid-like modules appear in both sets of receptors within key ligand-binding regions. Transmembrane regions associated with homodimerization of each class of receptors are also highly conserved across receptor types and implicated in heterodimerization. This conservation of multiple functional modules suggests opioid–adrenergic ligand and receptor co-evolution and provides mechanisms for explaining the evolution of their crosstalk. These modules also suggest the structure of a primordial receptor, providing clues for engineering receptor functions.

Microtubule dynamics influence the retrograde biased motility of kinesin-4 motor teams in neuronal dendrites

Microtubules establish the directionality of intracellular transport by kinesins and dynein through polarized assembly, but it remains unclear how directed transport occurs along microtubules organized with mixed polarity. We investigated the ability of the plus-end directed kinesin-4 motor KIF21B to navigate mixed polarity microtubules in mammalian dendrites. Reconstitution assays with recombinant KIF21B and engineered microtubule bundles or extracted neuronal cytoskeletons indicate that nucleotide-independent microtubule binding regions of KIF21B modulate microtubule dynamics and promote directional switching on antiparallel microtubules. Optogenetic recruitment of KIF21B to organelles in live neurons induces unidirectional transport in axons but bi-directional transport with a net retrograde bias in dendrites. Removal of the secondary microtubule binding regions of KIF21B or dampening of microtubule dynamics with low concentrations of nocodazole eliminates retrograde bias in live dendrites. Further exploration of the contribution of microtubule dynamics in dendrites to directionality revealed plus-end-out microtubules to be more dynamic than plus-end-in microtubules, with nocodazole preferentially stabilizing the plus-end-out population. We propose a model in which both nucleotide-sensitive and insensitive microtubule binding sites of KIF21B motors contribute to the search and selection of stable plus-end-in microtubules within the mixed polarity microtubule arrays characteristic of mammalian dendrites to achieve net retrograde movement of KIF21B-bound cargos. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Hypertrophic cardiomyopathy mutations in the pliant and light chain-binding regions of the lever arm of human β-cardiac myosin have divergent effects on myosin function

ABSTRACTMutations in the lever arm of β-cardiac myosin are a frequent cause of hypertrophic cardiomyopathy (HCM), a disease characterized by hypercontractility and eventual hypertrophy of the left ventricle. Here, we studied five such mutations: three in the pliant region of the lever arm (D778V, L781P, and S782N) and two in the light chain-binding region (A797T and F834L). We investigated their effects on both motor function and myosin S2 tail-based autoinhibition. The pliant region mutations had varying effects on the motor function of a myosin construct lacking the S2 tail: overall, D778V increased power output, L781P reduced power output, and S782N had little effect on power output, while all three reduced the external force sensitivity of the actin detachment rate. With a myosin containing the motor domain and the proximal S2 tail, the pliant region mutations also attenuated autoinhibition in the presence of filamentous actin but had no impact in the absence of actin. By contrast, the light chain-binding region mutations had little effect on motor activity but produced marked reductions in autoinhibition in both the presence and absence of actin. Thus, mutations in the lever arm of β-cardiac myosin have divergent allosteric effects on myosin function, depending on whether they are in the pliant or light chain-binding regions.

Ancestral protein topologies draw the rooted bacterial tree of life

Many experimental and predicted observations remain unanswered in the current proposed trees of life (ToL). Also, the current trend in reporting phylogenetic data is based in mixing together the information of dozens of genomes or entire conserved proteins. In this work, we consider the modularity of protein evolution and, using only two domains with duplicated ancestral topologies from a single, universal primordial protein corresponding to the RNA binding regions of contemporary bacterial glycyl tRNA synthetase (bacGlyRS), archaeal CCA adding enzyme (arch-CCAadd) and eukaryotic rRNA processing enzyme (euk-rRNA), we propose a rooted bacterial ToL that agrees with several previous observations unaccounted by the available trees.

ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals.

Ubiquitin (Ub) serves as a signal for clathrin-mediated endocytosis (CME) by engaging Ub-binding proteins with the internalization apparatus. Ub is a versatile internalization signal because it can be added to a wide variety of membrane proteins, expanding the capacity of cells to use a variety of regulatory mechanisms to specify the conditions under which a particular protein will be internalized. Several candidate adaptors that can recognize ubiquitinated membrane proteins have been identified that work in endocytic processes that are both clathrin-dependent and independent. These include Epsin and Eps15, which bind and help sort Ub-cargo into internalization sites. Here we identify additional components of the endocytosis apparatus that bind Ub. The N-terminal ANTH domains found in CALM, AP180, HIP1R and yeast Sla2 all bind monoubiquitin with micromolar affinity. ANTH domains belong to a larger superfamily of domains including ENTH and VHS domains, many of which have Ub-binding regions outside of their VHS/ENTH/ANTH domains that enable them to mediate Ub-dependent sorting events throughout the cell. Solution NMR studies combined with a crystal structure of the CALM ANTH domain in a complex with Ub show that Ub binds to a C-terminal region of the ANTH domain that is not present in ENTH domains. Combined loss of Ub-binding by ANTH-domain proteins and other Ub-binding domains within the internalization apparatus of yeast caused defects in the Ub-dependent internalization of the GPCR Ste2 but had no effect on internalization of Ste2 via other internalization signals. These studies define new components of the internalization machinery that work collectively with Epsin and Eps15 to specify recognition of Ub as an internalization signal.

Improved data sets and evaluation methods for the automatic prediction of DNA-binding proteins

Accurate automatic annotation of protein function relies on both innovative models and robust data sets. Due to their importance in biological processes, the identification of DNA-binding proteins directly from protein sequence has been the focus of many studies. However, the data sets used to train and evaluate these methods have suffered from substantial flaws. We describe some of the weaknesses of the data sets used in previous DNA-binding protein literature and provide several new data sets addressing these problems. We suggest new evaluative benchmark tasks that more realistically assess real-world performance for protein annotation models. We propose a simple new model for the prediction of DNA-binding proteins and compare its performance on the improved data sets to two previously published models. Additionally, we provide extensive tests showing how the best models predict across taxonomies. Our new gradient boosting model, which uses features derived from a published protein language model, outperforms the earlier models. Perhaps surprisingly, so does a baseline nearest neighbor model using BLAST percent identity. We evaluate the sensitivity of these models to perturbations of DNA-binding regions and control regions of protein sequences. The successful data-driven models learn to focus on DNA-binding regions. When predicting across taxonomies, the best models are highly accurate across species in the same kingdom and can provide some information when predicting across kingdoms.