Evidence for Non-Essential Salt Bridges in the M-Gates of Mitochondrial Carrier Proteins.

Mitochondrial carriers, which transport metabolites, nucleotides, and cofactors across the mitochondrial inner membrane, have six transmembrane α-helices enclosing a translocation pore with a central substrate binding site whose access is controlled by a cytoplasmic and a matrix gate (M-gate). The salt bridges formed by the three PX[DE]XX[RK] motifs located on the odd-numbered transmembrane α-helices greatly contribute to closing the M-gate. We have measured the transport rates of cysteine mutants of the charged residue positions in the PX[DE]XX[RK] motifs of the bovine oxoglutarate carrier, the yeast GTP/GDP carrier, and the yeast NAD+ transporter, which all lack one of these charged residues. Most single substitutions, including those of the non-charged and unpaired charged residues, completely inactivated transport. Double mutations of charged pairs showed that all three carriers contain salt bridges non-essential for activity. Two double substitutions of these non-essential charge pairs exhibited higher transport rates than their corresponding single mutants, whereas swapping the charged residues in these positions did not increase activity. The results demonstrate that some of the residues in the charged residue positions of the PX[DE]XX[KR] motifs are important for reasons other than forming salt bridges, probably for playing specific roles related to the substrate interaction-mediated conformational changes leading to the M-gate opening/closing.

New insights into the phosphorylation of the threonine motif of the ß1 integrin cytoplasmic domain.

Integrins require an activation step before ligand binding and signaling that is mediated by talin and kindlin binding to the ß integrin cytosolic domain (ß-tail). Conflicting reports exist about the contribution of phosphorylation of a conserved threonine motif in the ß1-tail (ß1-pT788/pT789) to integrin activation. We show that widely used and commercially available antibodies against ß1-pT788/pT789 integrin do not detect specific ß1-pT788/pT789 integrin signals in immunoblots of several human and mouse cell lysates but bind bi-phosphorylated threonine residues in numerous proteins, which were identified by mass spectrometry experiments. Furthermore, we found that fibroblasts and epithelial cells expressing the phospho-mimicking ß1-TT788/789DD integrin failed to activate ß1 integrins and displayed reduced integrin ligand binding, adhesion initiation and cell spreading. These cellular defects are specifically caused by the inability of kindlin to bind ß1-tail polypeptides carrying a phosphorylated threonine motif or phospho-mimicking TT788/789DD substitutions. Our findings indicate that the double-threonine motif in ß1-class integrins is not a major phosphorylation site but if phosphorylated would curb integrin function.

Helicase-like functions in phosphate loop containing beta-alpha polypeptides.

The P-loop Walker A motif underlies hundreds of essential enzyme families that bind nucleotide triphosphates (NTPs) and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/RNA helicases, translocases, and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple ßα repeat proteins bind NTPs but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one ß-(P-loop)-α element. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. The latter are reminiscent of extant P-loop recombinases such as RecA. Overall, these P-loop prototypes compose a plausible description of the sequence, structure, and function of the earliest P-loop NTPases. They also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.

Targeting nucleolin by RNA G-quadruplex-forming motif.

A high level of nucleolin (NCL) expression is often associated with a poor prognosis of patients with lung cancer (LC), suggesting that NCL can be used as a possible biomarker. NCL has been shown to display a marked preference for the binding to G-quadruplexes (G4). Here, we investigate the formation of an RNA quadruplex structure in a sequence found in the human precursor pre-MIR150 with the potential to recognize NCL. Circular dichroism (CD) spectra of pre-MIR150 G4-forming sequence (designated by rG4) indicate the formation of a parallel quadruplex structure in KCl or when complexed with the well-known G4 ligand PhenDC3. The thermal stability of rG4 is very high, and further increases in the presence of PhenDC3. The binding affinities of rG4 to PhenDC3 and NCL RBD1,2 are similar with KD values in the nanomolar range. PAGE results suggest the formation of a ternary quadruplex-ligand-protein complex (rG4-PhenDC3-NCL RBD1,2), indicative that PhenDC3 does not prevent the binding of rG4 to NCL RBD1,2. Finally, rG4 can recognize NCL-positive cells and, when fluorescently labeled, can be used as a probe for this protein. ELISA experiments indicate altered NCL expression patterns in liquid biopsies of LC patients in a non-invasive manner, potentially helping the diagnosis, prognosis, and patient response to treatment. Hence, labeled rG4 could be used as a detection probe of LC in liquid biopsies.

The Role of ICL1 and H8 in Class B1 GPCRs; Implications for Receptor Activation.

The first intracellular loop (ICL1) of G protein-coupled receptors (GPCRs) has received little attention, although there is evidence that, with the 8th helix (H8), it is involved in early conformational changes following receptor activation as well as contacting the G protein ß subunit. In class B1 GPCRs, the distal part of ICL1 contains a conserved R12.48KLRCxR2.46b motif that extends into the base of the second transmembrane helix; this is weakly conserved as a [R/H]12.48KL[R/H] motif in class A GPCRs. In the current study, the role of ICL1 and H8 in signaling through cAMP, iCa2+ and ERK1/2 has been examined in two class B1 GPCRs, using mutagenesis and molecular dynamics. Mutations throughout ICL1 can either enhance or disrupt cAMP production by CGRP at the CGRP receptor. Alanine mutagenesis identified subtle differences with regard elevation of iCa2+, with the distal end of the loop being particularly sensitive. ERK1/2 activation displayed little sensitivity to ICL1 mutation. A broadly similar pattern was observed with the glucagon receptor, although there were differences in significance of individual residues. Extending the study revealed that at the CRF1 receptor, an insertion in ICL1 switched signaling bias between iCa2+ and cAMP. Molecular dynamics suggested that changes in ICL1 altered the conformation of ICL2 and the H8/TM7 junction (ICL4). For H8, alanine mutagenesis showed the importance of E3908.49b for all three signal transduction pathways, for the CGRP receptor, but mutations of other residues largely just altered ERK1/2 activation. Thus, ICL1 may modulate GPCR bias via interactions with ICL2, ICL4 and the Gß subunit.

The C-terminal acidic motif of Phafin2 inhibits PH domain binding to phosphatidylinositol 3-phosphate.

Changes in membrane curvature are required to control the function of subcellular compartments; malfunctions of such processes are associated with a wide range of human diseases. Membrane remodeling often depends upon the presence of phosphoinositides, which recruit protein effectors for a variety of cellular functions. Phafin2 is a phosphatidylinositol 3-phosphate (PtdIns3P)-binding effector involved in endosomal and lysosomal membrane-associated signaling. Both the Phafin2 PH and the FYVE domains bind PtdIns3P, although their redundant function in the protein is unclear. Through a combination of lipid-binding assays, we found that, unlike the FYVE domain, recognition of the PH domain to PtdIns3P requires a lipid bilayer. Using site-directed mutagenesis and truncation constructs, we discovered that the Phafin2 FYVE domain is constitutive for PtdIns3P binding, whereas PH domain binding to PtdIns3P is autoinhibited by a conserved C-terminal acidic motif. These findings suggest that binding of the Phafin2 PH domain to PtdIns3P in membrane compartments occurs through a highly regulated mechanism. Potential mechanisms are discussed throughout this report.

A Disintegrin and Metalloproteinase (ADAM) and ADAM with thrombospondin motifs (ADAMTS) family in vascular biology and disease.

A Disintegrin and Metalloproteinase (ADAM) is a family of proteolytic enzymes that possess sheddase function and regulate shedding of membrane-bound proteins, growth factors, cytokines, ligands and receptors. Typically, ADAMs have a pro-domain, and a metalloproteinase, disintegrin, cysteine-rich and a characteristic transmembrane domain. Most ADAMs are activated by proprotein convertases, but can also be regulated by G-protein coupled receptor agonists, Ca2+ ionophores and protein kinase C activators. A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) is a family of secreted enzymes closely related to ADAMs. Like ADAMs, ADAMTS members have a pro-domain, and a metalloproteinase, disintegrin, and cysteine-rich domain, but they lack a transmembrane domain and instead have characteristic thrombospondin motifs. Activated ADAMs perform several functions and participate in multiple cardiovascular processes including vascular smooth muscle cell proliferation and migration, angiogenesis, vascular cell apoptosis, cell survival, tissue repair, and wound healing. ADAMs may also be involved in pathological conditions and cardiovascular diseases such as atherosclerosis, hypertension, aneurysm, coronary artery disease, myocardial infarction and heart failure. Like ADAMs, ADAMTS have a wide-spectrum role in vascular biology and cardiovascular pathophysiology. ADAMs and ADAMTS activity is naturally controlled by endogenous inhibitors such as tissue inhibitors of metalloproteinases (TIMPs), and their activity can also be suppressed by synthetic small molecule inhibitors. ADAMs and ADAMTS can serve as important diagnostic biomarkers and potential therapeutic targets for cardiovascular disorders. Natural and synthetic inhibitors of ADAMs and ADAMTS could be potential therapeutic tools for the management of cardiovascular diseases.

Overview of BCL-2 Family Proteins and Therapeutic Potentials.

BCL-2 family proteins interact in a network that regulates apoptosis. The BH3 amino acid sequence motif serves to bind together this conglomerate protein family, both literally and figuratively. BH3 motifs are present in antiapoptotic and proapoptotic BCL-2 homologs, and in a separate group of unrelated BH3-only proteins often appended to the BCL-2 family. BH3-containing helices mediate many of their physical interactions to determine cell death versus survival, leading to the development of BH3 mimetics as therapeutics. Here we provide an overview of BCL-2 family interactions, their relevance in health and disease, and the progress toward regulating their interactions therapeutically.

Regulation of the opposing (p)ppGpp synthetase and hydrolase activities in a bifunctional RelA/SpoT homologue from Staphylococcus aureus.

The stringent response is characterized by (p)ppGpp synthesis resulting in repression of translation and reprogramming of the transcriptome. In Staphylococcus aureus, (p)ppGpp is synthesized by the long RSH (RelA/SpoT homolog) enzyme, RelSau or by one of the two short synthetases (RelP, RelQ). RSH enzymes are characterized by an N-terminal enzymatic domain bearing distinct motifs for (p)ppGpp synthetase or hydrolase activity and a C-terminal regulatory domain (CTD) containing conserved motifs (TGS, DC and ACT). The intramolecular switch between synthetase and hydrolase activity of RelSau is crucial for the adaption of S. aureus to stress (stringent) or non-stress (relaxed) conditions. We elucidated the role of the CTD in the enzymatic activities of RelSau. Growth pattern, transcriptional analyses and in vitro assays yielded the following results: i) in vivo, under relaxed conditions, as well as in vitro, the CTD inhibits synthetase activity but is not required for hydrolase activity; ii) under stringent conditions, the CTD is essential for (p)ppGpp synthesis; iii) RelSau lacking the CTD exhibits net hydrolase activity when expressed in S. aureus but net (p)ppGpp synthetase activity when expressed in E. coli; iv) the TGS and DC motifs within the CTD are required for correct stringent response, whereas the ACT motif is dispensable, v) Co-immunoprecipitation indicated that the CTD interacts with the ribosome, which is largely dependent on the TGS motif. In conclusion, RelSau primarily exists in a synthetase-OFF/hydrolase-ON state, the TGS motif within the CTD is required to activate (p)ppGpp synthesis under stringent conditions.

LysM1 in MmLYK2 is a motif required for the interaction of MmLYP1 and MmLYK2 in the chitin signaling.

KEY MESSAGE: Two LysM-containing proteins, namely, MmLYP1 and MmLYK2, were identified in mulberry. These proteins might be involved in chitin signaling. The LysM1 of MmLYK2 is critical for their interactions. Chitin is a major component of fungal cell walls and acts as an elicitor in plant innate immunity. Lysin motif (LysM)-containing proteins are essential for chitin recognition. However, related studies have been rarely reported in woody plants. In this study, in mulberry, the expression of a LysM-containing protein, MmLYP1, was significantly up-regulated after treatment with chitin and pathogenic fungi. In addition, MmLYP1 has an affinity for insoluble chitin polymers. Thus, MmLYP1 might function in chitin signaling. Since MmLYP1 lacks an intracellular domain, additional protein kinases are required for this signaling. An LysM-containing kinase, MmLYK2, was then identified. Expression of the MmLYK2 did not change significantly after chitin treatment, and the affinity of MmLYK2 for insoluble chitin was not high. The structure of MmLYP1 is similar to that of the chitin elicitor-binding proteins in rice and Arabidopsis. However, MmLYK2 has two LysM motifs, while the chitin elicitor receptor kinase 1 proteins in rice and Arabidopsis have one and three LysM motifs, respectively. The LysM1 of MmLYK2 interacted with all four LysM motifs in MmLYP1 and MmLYK2 in yeast. The chimera lacking the LysM1 of MmLYK2 did not interact with MmLYP1 and MmLYK2 in yeast and Nicotiana benthamiana cells. The LysM1 in MmLYK2 is the key motif in the interaction between MmLYP1 and MmLYK2, which may be involved in chitin signaling.

VLITL is a major cross-ß-sheet signal for fibrinogen Aα-chain frameshift variants.

The first case of hereditary fibrinogen Aα-chain amyloidosis was recognized >20 years ago, but disease mechanisms still remain unknown. Here we report detailed clinical and proteomics studies of a French kindred with a novel amyloidogenic fibrinogen Aα-chain frameshift variant, Phe521Leufs, causing a severe familial form of renal amyloidosis. Next, we focused our investigations to elucidate the molecular basis that render this Aα-chain variant amyloidogenic. We show that a 49-mer peptide derived from the C-terminal part of the Phe521Leufs chain is deposited as fibrils in the patient's kidneys, establishing that only a small portion of Phe521Leufs directly contributes to amyloid formation in vivo. In silico analysis indicated that this 49-mer Aα-chain peptide contained a motif (VLITL), with a high intrinsic propensity for ß-aggregation at residues 44 to 48 of human renal fibrils. To experimentally verify the amyloid propensity of VLITL, we generated synthetic Phe521Leufs-derived peptides and compared their capacity for fibril formation in vitro with that of their VLITL-deleted counterparts. We show that VLITL forms typical amyloid fibrils in vitro and is a major signal for cross-ß-sheet self-association of the 49-mer Phe521Leufs peptide identified in vivo, whereas its absence abrogates fibril formation. This study provides compelling evidence that VLITL confers amyloidogenic properties to Aα-chain frameshift variants, yielding a previously unknown molecular basis for the pathogenesis of Aα-chain amyloidosis.

An N-terminal di-proline motif is essential for fatty acid-dependent degradation of Δ9-desaturase in Drosophila.

The Δ9-fatty acid desaturase introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA and regulates the cellular levels of unsaturated fatty acids. However, it is unclear how Δ9-desaturase expression is regulated in response to changes in the levels of fatty acid desaturation. In this study, we found that the degradation of DESAT1, the sole Δ9-desaturase in the Drosophila cell line S2, was significantly enhanced when the amounts of unsaturated acyl chains of membrane phospholipids were increased by supplementation with unsaturated fatty acids, such as oleic and linoleic acids. In contrast, inhibition of DESAT1 activity remarkably suppressed its degradation. Of note, removal of the DESAT1 N-terminal domain abolished the responsiveness of DESAT1 degradation to the level of fatty acid unsaturation. Further truncation and amino acid replacement analyses revealed that two sequential prolines, the second and third residues of DESAT1, were responsible for the unsaturated fatty acid-dependent degradation. Although degradation of mouse stearoyl-CoA desaturase 1 (SCD1) was unaffected by changes in fatty acid unsaturation, introduction of the N-terminal sequential proline residues into SCD1 conferred responsiveness to unsaturated fatty acid-dependent degradation. Furthermore, we also found that the Ca2+-dependent cysteine protease calpain is involved in the sequential proline-dependent degradation of DESAT1. In light of these findings, we designated the sequential prolines at the second and third positions of DESAT1 as a "di-proline motif," which plays a crucial role in the regulation of Δ9-desaturase expression in response to changes in the level of cellular unsaturated fatty acids.

IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole.

Membrane contact sites (MCS) are zones of contact between the membranes of two organelles. At MCS, specific proteins tether the organelles in close proximity and mediate the nonvesicular trafficking of lipids and ions between the two organelles. The endoplasmic reticulum (ER) integral membrane protein VAP is a common component of MCS involved in both tethering and lipid transfer by binding directly to proteins containing a FFAT [two phenylalanines (FF) in an acidic tract (AT)] motif. In addition to maintaining cell homeostasis, MCS formation recently emerged as a mechanism by which intracellular pathogens hijack cellular resources and establish their replication niche. Here, we investigated the mechanism by which the Chlamydia-containing vacuole, termed the inclusion, establishes direct contact with the ER. We show that the Chlamydia protein IncV, which is inserted into the inclusion membrane, displays one canonical and one noncanonical FFAT motif that cooperatively mediated the interaction of IncV with VAP. IncV overexpression was sufficient to bring the ER in close proximity of IncV-containing membranes. Although IncV deletion partially decreased VAP association with the inclusion, it did not suppress the formation of ER-inclusion MCS, suggesting the existence of redundant mechanisms in MCS formation. We propose a model in which IncV acts as one of the primary tethers that contribute to the formation of ER-inclusion MCS. Our results highlight a previously unidentified mechanism of bacterial pathogenesis and support the notion that cooperation of two FFAT motifs may be a common feature of VAP-mediated MCS formation. Chlamydia-host cell interaction therefore constitutes a unique system to decipher the molecular mechanisms underlying MCS formation.

Global mapping of CARM1 substrates defines enzyme specificity and substrate recognition.

Protein arginine methyltransferases (PRMTs) introduce arginine methylation, a post-translational modification with the increasingly eminent role in normal physiology and disease. PRMT4 or coactivator-associated arginine methyltransferase 1 (CARM1) is a propitious target for cancer therapy; however, few CARM1 substrates are known, and its mechanism of substrate recognition is poorly understood. Here we employed a quantitative mass spectrometry approach to globally profile CARM1 substrates in breast cancer cell lines. We identified >130 CARM1 protein substrates and validated in vitro >90% of sites they encompass. Bioinformatics analyses reveal enrichment of proline-containing motifs, in which both methylation sites and their proximal sequences are frequently targeted by somatic mutations in cancer. Finally, we demonstrate that the N-terminus of CARM1 is involved in substrate recognition and nearly indispensable for substrate methylation. We propose that development of CARM1-specific inhibitors should focus on its N-terminus and predict that other PRMTs may employ similar mechanism for substrate recognition.

Comparison of extracellular Cys/Trp motif between Schizosaccharomyces pombe Ctr4 and Ctr5.

The reduction and binding of copper ions to the Cys/Trp motif, which is characterized by two cysteines and two tryptophans, in the extracellular N-terminal domain of the copper transporter (Ctr) protein of fungi are investigated using the model peptides of Ctr4 and Ctr5 from Schizosaccharomyces pombe. The Cys/Trp motif of Ctr5 can reduce Cu(II) and ligate Cu(I), which is the same as that of Ctr4 previously reported. Titration of Cu(II) and Cu(I) ions indicates that both the Cys/Trp motifs of Ctr4 and Ctr5 reduce two Cu(II) and bind two Cu(I) per one peptide. However, the coordination structure of the Cu(I)-peptide complex differs between Ctr4 and Ctr5. Cu(I) is bound to the Cys/Trp motif of Ctr5 via cysteine thiolate-Cu(I) bonds and cation-π interaction with tryptophan, as reported for Ctr4, and a histidine residue in the Cys/Trp motif of Ctr5 is suggested to interact with Cu(I) via its Nτ atom. Ctr4 and Ctr5 exhibit a heterotrimeric form within cell membranes and the copper transport mechanism of the Ctr4/Ctr5 heterotrimer is discussed along with quantitative evaluation of the Cu(I)-binding constant of the Cys/Trp motif.

Nucleotide binding by the widespread high-affinity cyclic di-GMP receptor MshEN domain.

C-di-GMP is a bacterial second messenger regulating various cellular functions. Many bacteria contain c-di-GMP-metabolizing enzymes but lack known c-di-GMP receptors. Recently, two MshE-type ATPases associated with bacterial type II secretion system and type IV pilus formation were shown to specifically bind c-di-GMP. Here we report crystal structure of the MshE N-terminal domain (MshEN1-145) from Vibrio cholerae in complex with c-di-GMP at a 1.37 Å resolution. This structure reveals a unique c-di-GMP-binding mode, featuring a tandem array of two highly conserved binding motifs, each comprising a 24-residue sequence RLGxx(L/V/I)(L/V/I)xxG(L/V/I)(L/V/I)xxxxLxxxLxxQ that binds half of the c-di-GMP molecule, primarily through hydrophobic interactions. Mutating these highly conserved residues markedly reduces c-di-GMP binding and biofilm formation by V. cholerae. This c-di-GMP-binding motif is present in diverse bacterial proteins exhibiting binding affinities ranging from 0.5 µM to as low as 14 nM. The MshEN domain contains the longest nucleotide-binding motif reported to date.

Histone Sprocket Arginine Residues Are Important for Gene Expression, DNA Repair, and Cell Viability in Saccharomyces cerevisiae.

A critical feature of the intermolecular contacts that bind DNA to the histone octamer is the series of histone arginine residues that insert into the DNA minor groove at each superhelical location where the minor groove faces the histone octamer. One of these "sprocket" arginine residues, histone H4 R45, significantly affects chromatin structure in vivo and is lethal when mutated to alanine or cysteine in Saccharomyces cerevisiae (budding yeast). However, the roles of the remaining sprocket arginine residues (H3 R63, H3 R83, H2A R43, H2B R36, H2A R78, H3 R49) in chromatin structure and other cellular processes have not been well characterized. We have genetically characterized mutations in each of these histone residues when introduced either singly or in combination to yeast cells. We find that pairs of arginine residues that bind DNA adjacent to the DNA exit/entry sites in the nucleosome are lethal in yeast when mutated in combination and cause a defect in histone occupancy. Furthermore, mutations in individual residues compromise repair of UV-induced DNA lesions and affect gene expression and cryptic transcription. This study reveals simple rules for how the location and structural mode of DNA binding influence the biological function of each histone sprocket arginine residue.

Alternative Protein Secretion in the Malaria Parasite Plasmodium falciparum.

Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacuole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of N-myristoylation and two other N-terminal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM) and subsequent translocation across this membrane. Plasmodium falciparum adenylate kinase 2 (Pf AK2) contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of PfAK2 variants tagged with green fluorescent protein (GFP) showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-terminal 37 amino acids of PfAK2 alone are sufficient to target and translocate GFP across the PPM. As a control we examined the N-myristoylated P. falciparum ADP-ribosylation factor 1 (PfARF1). PfARF1 was found to co-localise with a Golgi marker. To determine whether or not the putative palmitoylation and the cluster of lysine residues from the N-terminus of PfAK2 would modulate the subcellular localization of PfARF1, a chimeric fusion protein containing the N-terminus of PfARF1 and the two additional PfAK2 motifs was analysed. This chimeric protein was targeted to the PPM, but not translocated across the membrane into the PV, indicating that other features of the N-terminus of PfAK2 also play a role in the secretion process.

DYNLL2 dynein light chain binds to an extended linear motif of myosin 5a tail that has structural plasticity.

LC8 dynein light chains (DYNLL) are conserved homodimeric eukaryotic hub proteins that participate in diverse cellular processes. Among the binding partners of DYNLL2, myosin 5a (myo5a) is a motor protein involved in cargo transport. Here we provide a profound characterization of the DYNLL2 binding motif of myo5a in free and DYNLL2-bound form by using nuclear magnetic resonance spectroscopy, X-ray crystallography, and molecular dynamics simulations. In the free form, the DYNLL2 binding region, located in an intrinsically disordered domain of the myo5a tail, has a nascent helical character. The motif becomes structured and folds into a ß-strand upon binding to DYNLL2. Despite differences of the myo5a sequence from the consensus binding motif, one peptide is accommodated in each of the parallel DYNLL2 binding grooves, as for all other known partners. Interestingly, while the core motif shows a similar interaction pattern in the binding groove as seen in other complexes, the flanking residues make several additional contacts, thereby lengthening the binding motif. The N-terminal extension folds back and partially blocks the free edge of the ß-sheet formed by the binding motif itself. The C-terminal extension contacts the dimer interface and interacts with symmetry-related residues of the second myo5a peptide. The involvement of flanking residues of the core binding site of myo5a could modify the quaternary structure of the full-length myo5a and affect its biological functions. Our results deepen the knowledge of the diverse partner recognition of DYNLL proteins and provide an example of a Janus-faced linear motif.

Acetylation-defective mutant of Pparγ is associated with decreased lipid synthesis in breast cancer cells.

In our prior publications we characterized a conserved acetylation motif (K(R)xxKK) of evolutionarily related nuclear receptors. Recent reports showed that peroxisome proliferator activated receptor gamma (PPARγ) deacetylation by SIRT1 is involved in delaying cellular senescence and maintaining the brown remodeling of white adipose tissue. However, it still remains unknown whether lysyl residues 154 and 155 (K154/155) of the conserved acetylation motif (RIHKK) in Pparγ1 are acetylated. Herein, we demonstrate that Pparγ1 is acetylated and regulated by both endogenous TSA-sensitive and NAD-dependent deacetylases. Acetylation of lysine 154 was identified by mass spectrometry (MS) while deacetylation of lysine 155 by SIRT1 was confirmed by in vitro deacetylation assay. An in vivo labeling assay revealed K154/K155 as bona fide acetylation sites. The conserved acetylation sites of Pparγ1 and the catalytic domain of SIRT1 are both required for the interaction between Pparγ1 and SIRT1. Sirt1 and Pparγ1 converge to govern lipid metabolism in vivo. Acetylation-defective mutants of Pparγ1 were associated with reduced lipid synthesis in ErbB2 overexpressing breast cancer cells. Together, these results suggest that the conserved lysyl residues K154/K155 of Pparγ1 are acetylated and play an important role in lipid synthesis in ErbB2-positive breast cancer cells.