Structural dynamics in the evolution of a bilobed protein scaffold

Novel biophysical tools allow the structural dynamics of proteins and the regulation of such dynamics by binding partners to be explored in unprecedented detail. Although this has provided critical insights into protein function, the means by which structural dynamics direct protein evolution remain poorly understood. Here, we investigated how proteins with a bilobed structure, composed of two related domains from the periplasmic-binding protein–like II domain family, have undergone divergent evolution, leading to adaptation of their structural dynamics. We performed a structural analysis on ∼600 bilobed proteins with a common primordial structural core, which we complemented with biophysical studies to explore the structural dynamics of selected examples by single-molecule Förster resonance energy transfer and Hydrogen–Deuterium exchange mass spectrometry. We show that evolutionary modifications of the structural core, largely at its termini, enable distinct structural dynamics, allowing the diversification of these proteins into transcription factors, enzymes, and extracytoplasmic transport-related proteins. Structural embellishments of the core created interdomain interactions that stabilized structural states, reshaping the active site geometry, and ultimately altered substrate specificity. Our findings reveal an as-yet-unrecognized mechanism for the emergence of functional promiscuity during long periods of evolution and are applicable to a large number of domain architectures.

Reciprocal allostery arising from a bi-enzyme assembly controls aromatic amino acid biosynthesis in Prevotella nigrescens

ABSTRACTModular protein assembly has been widely reported as a mechanism for constructing allosteric machinery. Recently, a distinctive allosteric system has been identified in a bi-enzyme assembly comprising a 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM). These enzymes catalyze the first and branch point reactions of aromatic amino acid biosynthesis in the bacterium Prevotella nigrescens (PniDAH7PS), respectively. The interactions between these two distinct catalytic domains support functional inter-reliance within this bifunctional enzyme. The binding of prephenate, the product of CM-catalyzed reaction, to the CM domain is associated with a striking rearrangement of overall protein conformation that alters the interdomain interactions and allosterically inhibits the DAH7PS activity. In this study, we observed allosteric activation of CM activity in the presence of all DAH7PS substrates. Using small angle X-ray scattering (SAXS) experiments we show that changes in overall protein conformations and dynamics are associated with the presence of different DAH7PS substrates and the allosteric inhibitor prephenate. Furthermore, we have identified an extended interhelix loop located in CM domain, loopC320-F333, as a crucial segment for the interdomain structural and catalytic communications. Our results suggest that the dual function enzyme PniDAH7PS contains a reciprocal allosteric system between the two enzymatic moieties, as a result of this bidirectional interdomain communication. This arrangement allows for a complex feedback and feedforward system for control of pathway flux by connecting the initiation and branch point of aromatic amino acid biosynthesis.

Interdomain interactions regulate the localization of a lipid transfer protein at ER-PM contact sites

ABSTRACT During phospholipase C-β (PLC-β) signalling in Drosophila photoreceptors, the phosphatidylinositol transfer protein (PITP) RDGB, is required for lipid transfer at endoplasmic reticulum (ER)–plasma membrane (PM) contact sites (MCS). Depletion of RDGB or its mis-localization away from the ER–PM MCS results in multiple defects in photoreceptor function. Previously, the interaction between the FFAT motif of RDGB and the integral ER protein dVAP-A was shown to be essential for accurate localization to ER–PM MCS. Here, we report that the FFAT/dVAP-A interaction alone is insufficient to localize RDGB accurately; this also requires the function of the C-terminal domains, DDHD and LNS2. Mutations in each of these domains results in mis-localization of RDGB leading to loss of function. While the LNS2 domain is necessary, it is not sufficient for the correct localization of RDGB, which also requires the C-terminal DDHD domain. The function of the DDHD domain is mediated through an intramolecular interaction with the LNS2 domain. Thus, interactions between the additional domains in a multi-domain PITP together lead to accurate localization at the MCS and signalling function. This article has an associated First Person interview with the first author of the paper.

First person – Bishal Basak

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Bishal Basak is first author on ‘Interdomain interactions regulate the localization of a lipid transfer protein at ER-PM contact sites’, published in BiO. Bishal is a PhD student in the lab of Professor Raghu Padinjat at National Center for Biological Sciences, Rajiv Gandhi Nagar, Kodigehalli, Bengaluru, Karnataka, India, investigating non-vesicular trafficking of lipids at interorganeller contact sites regulate cellular physiology.

Interdomain interactions regulate the localization of a lipid transfer protein at ER-PM contact sites

During phospholipase C-β (PLC-β) signalling in Drosophila photoreceptors, the phosphatidylinositol transfer protein (PITP) RDGB, is required for lipid transfer at endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (MCS). Depletion of RDGB or its mis-localization away from the ER-PM MCS results in multiple defects in photoreceptor function. Previously, the interaction between the FFAT motif of RDGB and the integral ER protein dVAP-A was shown to be essential for accurate localization to ER-PM MCS. Here, we report that the FFAT/dVAP-A interaction alone is insufficient to localize RDGB accurately; this also requires the function of the C-terminal domains, DDHD and LNS2. Mutations in each of these domains results in mis-localization of RDGB leading to loss of function. While the LNS2 domain is necessary, it is not sufficient for the correct localization of RDGB, which also requires the C-terminal DDHD domain. The function of the DDHD domain is mediated through an intramolecular interaction with the LNS2 domain. Thus, interactions between the additional domains in a multi-domain PITP together lead to accurate localization at the MCS and signalling function.

Coupled intra- and interdomain dynamics support domain cross-talk in Pin1

The functional mechanisms of multidomain proteins often exploit interdomain interactions, or “cross-talk.” An example is human Pin1, an essential mitotic regulator consisting of a Trp–Trp (WW) domain flexibly tethered to a peptidyl-prolyl isomerase (PPIase) domain, resulting in interdomain interactions important for Pin1 function. Substrate binding to the WW domain alters its transient contacts with the PPIase domain via means that are only partially understood. Accordingly, we have investigated Pin1 interdomain interactions using NMR paramagnetic relaxation enhancement (PRE) and molecular dynamics (MD) simulations. The PREs show that apo-Pin1 samples interdomain contacts beyond the range suggested by previous structural studies. They further show that substrate binding to the WW domain simultaneously alters interdomain separation and the internal conformation of the WW domain. A 4.5-μs all-atom MD simulation of apo-Pin1 suggests that the fluctuations of interdomain distances are correlated with fluctuations of WW domain interresidue contacts involved in substrate binding. Thus, the interdomain/WW domain conformations sampled by apo-Pin1 may already include a range of conformations appropriate for binding Pin1's numerous substrates. The proposed coupling between intra-/interdomain conformational fluctuations is a consequence of the dynamic modular architecture of Pin1. Such modular architecture is common among cell-cycle proteins; thus, the WW–PPIase domain cross-talk mechanisms of Pin1 may be relevant for their mechanisms as well.

Crystal Structure of Exotoxin A from Aeromonas Pathogenic Species

Aeromonas exotoxin A (AE) is a bacterial virulence factor recently discovered in a clinical case of necrotising fasciitis caused by the flesh-eating Aeromonas hydrophila. Here, database mining shows that AE is present in the genome of several emerging Aeromonas pathogenic species. The X-ray crystal structure of AE was solved at 2.3 Å and presents all the hallmarks common to diphthamide-specific mono-ADP-ribosylating toxins, suggesting AE is a fourth member of this family alongside the diphtheria toxin, Pseudomonas exotoxin A and cholix. Structural homology indicates AE may use a similar mechanism of cytotoxicity that targets eukaryotic elongation factor 2 and thus inhibition of protein synthesis. The structure of AE also highlights unique features including a metal binding site, and a negatively charged cleft that could play a role in interdomain interactions and may affect toxicity. This study raises new opportunities to engineer alternative toxin-based molecules with pharmaceutical potential.

Insights into channel modulation mechanism of RYR1 mutants using Ca2+ imaging and molecular dynamics

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum in skeletal muscle and plays an important role in excitation–contraction coupling. Mutations in the RYR1 gene cause severe muscle diseases such as malignant hyperthermia (MH), which is a disorder of CICR via RYR1. Thus far, >300 mutations in RYR1 have been reported in patients with MH. However, owing to a lack of comprehensive analysis of the structure–function relationship of mutant RYR1, the mechanism remains largely unknown. Here, we combined functional studies and molecular dynamics (MD) simulations of RYR1 bearing disease-associated mutations at the N-terminal region. When expressed in HEK293 cells, the mutant RYR1 caused abnormalities in Ca2+ homeostasis. MD simulations of WT and mutant RYR1s were performed using crystal structure of the N-terminal domain (NTD) monomer, consisting of A, B, and C domains. We found that the mutations located around the interdomain region differentially affected hydrogen bonds/salt bridges. Particularly, mutations at R402, which increase the open probability of the channel, cause clockwise rotation of BC domains with respect to the A domain by alteration of the interdomain interactions. Similar results were also obtained with artificial mutations that mimic alteration of the interactions. Our results reveal the importance of interdomain interactions within the NTD in the regulation of the RYR1 channel and provide insights into the mechanism of MH caused by the mutations at the NTD.

The lipid transfer function of RDGB at ER-PM contact sites is regulated by multiple interdomain interactions

SummaryIn Drosophila photoreceptors, following Phospholipase C-β activation, the phosphatidylinositol transfer protein (PITP) RDGB, is required to maintain lipid homeostasis at endoplasmic reticulum (ER) plasma membrane (PM) membrane contact sites (MCS). Depletion or mis-localization of RDGB results in multiple defects in photoreceptors. Previously, interaction between the FFAT motif of RDGB with the integral ER protein dVAP-A was shown to be important for its localization at ER-PM MCS. Here, we report that in addition to FFAT motif, a large unstructured region (USR1) of RDGB is required to support the RDGB/dVAP-A interaction. However, interaction with dVAP-A alone is insufficient for accurate localization of RDGB: this also requires association of RDGB with apical PM, through its C-terminal LNS2 domain. Deletion of LNS2 domain results in complete mis-localisation of RDGB and also induces large mis-regulated interdomain movements abrogating RDGB function. Thus, multiple independent interactions between individual domains of RDGB supports its function at ER-PM MCS.