Atomistic Structure and Dynamics of the Ca2+-ATPase Bound to Phosphorylated Phospholamban

Sarcoplasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLB) are essential components of the cardiac Ca2+ transport machinery. PLB phosphorylation at residue Ser16 (pSer16) enhances SERCA activity in the heart via an unknown structural mechanism. Here, we report a fully atomistic model of SERCA bound to phosphorylated PLB and study its structural dynamics on the microsecond time scale using all-atom molecular dynamics simulations in an explicit lipid bilayer and water environment. The unstructured N-terminal phosphorylation domain of PLB samples different orientations and covers a broad area of the cytosolic domain of SERCA but forms a stable complex mediated by pSer16 interactions with a binding site formed by SERCA residues Arg324/Lys328. PLB phosphorylation does not affect the interaction between the transmembrane regions of the two proteins; however, pSer16 stabilizes a disordered structure of the N-terminal phosphorylation domain that releases key inhibitory contacts between SERCA and PLB. We found that PLB phosphorylation is sufficient to guide the structural transitions of the cytosolic headpiece that are required to produce a competent structure of SERCA. We conclude that PLB phosphorylation serves as an allosteric molecular switch that releases inhibitory contacts and strings together the catalytic elements required for SERCA activation. This atomistic model represents a vivid atomic-resolution visualization of SERCA bound to phosphorylated PLB and provides previously inaccessible insights into the structural mechanism by which PLB phosphorylation releases SERCA inhibition in the heart.

Cryo-EM structures capture the transport cycle of the P4-ATPase flippase

In eukaryotic membranes, type IV P-type adenosine triphosphatases (P4-ATPases) mediate the translocation of phospholipids from the outer to the inner leaflet and maintain lipid asymmetry, which is critical for membrane trafficking and signaling pathways. Here, we report the cryo–electron microscopy structures of six distinct intermediates of the human ATP8A1-CDC50a heterocomplex at resolutions of 2.6 to 3.3 angstroms, elucidating the lipid translocation cycle of this P4-ATPase. ATP-dependent phosphorylation induces a large rotational movement of the actuator domain around the phosphorylation site in the phosphorylation domain, accompanied by lateral shifts of the first and second transmembrane helices, thereby allowing phosphatidylserine binding. The phospholipid head group passes through the hydrophilic cleft, while the acyl chain is exposed toward the lipid environment. These findings advance our understanding of the flippase mechanism and the disease-associated mutants of P4-ATPases.

Definition of the Minimal Truncated Elements Necessary for CSF3R Leukemogenic Potential

An increasing number of CSF3R cytoplasmic domain truncation (nonsense and frameshift) and missense mutations are being identified with the wide application of next generation sequencing (NGS) to leukemia patient samples. Previous studies have demonstrated oncogenic potential of some CSF3R truncation mutations seen in severe congenital neutropenia, chronic neutrophilia leukemia (CNL), and Philadelphia-negative atypical chronic neutrophilic leukemia (aCML) patients. However, full understanding of the spectrum of CSF3R cytoplasmic mutations harboring leukemogenic potential as well as the associated mechanisms of enhanced signaling remains incomplete. In the current study, we leveraged NGS data to first validate that CSF3R cytoplasmic mutations are prevalent in CNL/aCML/unclassified myeloproliferative neoplasm (8.5%, n=212) patients and to a lesser extent in AML patients (1.3%, n=378). We did not observe CSF3R cytoplasmic mutations in other hematologic malignances. Notably, 72.2% of CSF3R cytoplasmic mutations co-occur with the membrane proximal mutation, CSF3R T618I. To evaluate the leukemogenic potential of these cytoplasmic mutations, we first investigated the minimal mutated elements necessary for CSF3R leukemogenic potential. We identified that truncation mutations of the CSF3R cytoplasmic tail between T738 and Q823 all harbor leukemogenic potential. In contrast, CSF3R truncations before E700X did not respond to G-CSF and did not transform Ba/F3 cells even when combined with T618I. Truncation mutations between E700X and T738X demonstrated reduced G-CSF sensitivity, but could transform Ba/F3 cells when combined with CSF3R T618I. Surprisingly, all the cytoplasmic missense mutations (Q754A, R769H, L777F, T781I, S795R and Q823H), seen in the patient samples did not harbor leukemogenic potential. We further identified that missense mutations disrupting the di-leucine residues L776/777, S772, or ubiquitin binding lysine residues (K655, K785 and K704) demonstrated transforming capacity. Mechanistically, truncation mutations prior to Q793 are associated with delayed receptor internalization due to the disruption of the internalization motifs (amino acids 772-778 and 779-792), whereas truncation mutations between Q793 and Q823 decrease receptor degradation, which may be associated with loss of the de-phosphorylation domain (amino acids N818-F836). All truncation mutations before Q823 demonstrate altered patterns of molecular weight bands and are sensitive to the Golgi inhibitor brefeldin A, indicating that CSF3R glycosylation plays a role in the leukemogenic potential of these truncations. We also showed that K704 is a potential ubiquitin binding site, and substitution of K704A demonstrated reduced ubiquitination. Downstream, leukemogenic cytoplasmic mutations all induce sustained STAT5 activation and are sensitive to the JAK inhibitor ruxolitinib. In summary, we have defined the region of the CSF3R cytoplasmic domain in which truncating mutations exhibit the capacity for leukemogenesis. This information will be useful for evaluating the potential clinical relevance of CSF3R mutations appearing in genomic reports from patients with myeloid leukemia. Schematic illustration of CSF3R truncation mutations and the functional consequences. CSF3R truncation mutations at 658-685 which abrogate STAT5 activation do not transform BaF3 cells and do not respond to G-CSF, even when combined with T618I mutation. Truncation mutations at 700-738 do not transform Ba/F3 cells alone, however, these truncations can transform Ba/F3 when combined with T618I and are hyposensitive to G-CSF due to the disruption of the mitogenic enhancing domain. Truncation mutations between 738-791 and 793-823 transform Ba/F3 and induce G-CSF hypersensitivity due to the interruption of the internalization and the de-phosphorylation domain respectively. MED: mitogenic domain; D: Domain. Schematic illustration of CSF3R truncation mutations and the functional consequences. CSF3R truncation mutations at 658-685 which abrogate STAT5 activation do not transform BaF3 cells and do not respond to G-CSF, even when combined with T618I mutation. Truncation mutations at 700-738 do not transform Ba/F3 cells alone, however, these truncations can transform Ba/F3 when combined with T618I and are hyposensitive to G-CSF due to the disruption of the mitogenic enhancing domain. Truncation mutations between 738-791 and 793-823 transform Ba/F3 and induce G-CSF hypersensitivity due to the interruption of the internalization and the de-phosphorylation domain respectively. MED: mitogenic domain; D: Domain. Disclosures No relevant conflicts of interest to declare.

Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting

The actin filament severing protein cofilin-1 (CFL-1) is required for actin and P-type ATPase secretory pathway calcium ATPase (SPCA)-dependent sorting of secretory proteins at the trans-Golgi network (TGN). How these proteins interact and activate the pump to facilitate cargo sorting, however, is not known. We used purified proteins to assess interaction of the cytoplasmic domains of SPCA1 with actin and CFL-1. A 132–amino acid portion of the SPCA1 phosphorylation domain (P-domain) interacted with actin in a CFL-1–dependent manner. This domain, coupled to nickel nitrilotriacetic acid (Ni-NTA) agarose beads, specifically recruited F-actin in the presence of CFL-1 and, when expressed in HeLa cells, inhibited Ca2+ entry into the TGN and secretory cargo sorting. Mutagenesis of four amino acids in SPCA1 that represent the CFL-1 binding site also affected Ca2+ import into the TGN and secretory cargo sorting. Altogether, our findings reveal the mechanism of CFL-1–dependent recruitment of actin to SPCA1 and the significance of this interaction for Ca2+ influx and secretory cargo sorting.

Domains of the cucumber mosaic virus 2b silencing suppressor protein affecting inhibition of salicylic acid-induced resistance and priming of salicylic acid accumulation during infection

The cucumber mosaic virus (CMV) 2b silencing suppressor protein allows the virus to overcome resistance to replication and local movement in inoculated leaves of plants treated with salicylic acid (SA), a resistance-inducing plant hormone. In Arabidopsis thaliana plants systemically infected with CMV, the 2b protein also primes the induction of SA biosynthesis during this compatible interaction. We found that CMV infection of susceptible tobacco (Nicotiana tabacum) also induced SA accumulation. Utilization of mutant 2b proteins expressed during infection of tobacco showed that the N- and C-terminal domains, which had previously been implicated in regulation of symptom induction, were both required for subversion of SA-induced resistance, while all mutants tested except those affecting the putative phosphorylation domain had lost the ability to prime SA accumulation and expression of the SA-induced marker gene PR-1.