Flanking Residues Research Articles

Molecular mechanism for accurate dephosphorylation of RNA polymerase II during eukaryotic transcription

The phosphorylation states of the C‐terminal domain of RNA polymerase II (CTD) coordinate eukaryotic transcription by recruiting protein regulators to Pol II. The precise placement and removal of phosphate groups on the specific residues of the heptad repeat of CTD is critical for the fidelity and effectiveness of transcription. In spite of highly similar flanking residues, Ser5 dephosphorylation is carried out efficiently during transcription elongation when Ser2 phosphorylation accumulates, while Ser2 dephosphorylation occurs predominantly at the end of transcription. To understand the molecular mechanisms of how the CTD phosphatases Ssu72 and Fcp1 specifically remove CTD phosphorylation marks in the context of other phosphorylated CTD residues, we used a combination of high resolution MALDI‐TOF mass spectrometry, X‐ray crystallography, and protein engineering to analyze CTD phosphoregulation. Biochemical and bioanalytical characterization revealed that Ssu72 dephosphorylates Ser5 effectively but with much less activity towards Ser7 and Ser2 and no activity against Tyr1 and Thr4. Structural analysis demonstrated that the requirement for cis‐proline substrates of Ssu72 inhibits binding of bulky flanking residues like Tyr1 next to Ser2, which illustrates why Ser5 is the preferred substrate for Ssu72. Phosphorylation of Tyr1 even further prohibits binding of pSer2 by Ssu72, preventing the untimely dephosphorylation of Ser2 at elongation by Ssu72. Finally, we show that Fcp1 not only dephosphorylates phosphorylated Ser2 and Ser5 but also phosphorylated Tyr1. General clearance of phosphorylation marks on the CTD by Fcp1 supports its reported role in recycling Pol II for the next round of transcription. Our results reveal novel roles for Tyr1 in preventing inappropriate dephosphorylation of Ser2 by Ssu72 during transcriptional elongation and for Fcp1 in removing Tyr1 phosphorylation marks at the end of transcription.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Membrane Protein Integration and Topogenesis at the ER.

Most membrane proteins are composed of hydrophobic α-helical transmembrane segments and are integrated into the lipid bilayer of the endoplasmic reticulum by the highly conserved Sec61 translocon. With respect to the integration mechanism, three types of transmembrane segments can be distinguished-the signal, the stop-transfer sequence, and the re-integration sequence-which in linear succession can account for all kinds of membrane protein topologies. The transmembrane orientation of the initial signal and to a weaker extent also of downstream transmembrane segments is affected by charged flanking residues according to the so-called positive-inside rule. The main driving force for transmembrane integration is hydrophobicity. Systematic analysis suggested thermodynamic equilibration of each peptide segment in the translocon with the membrane as the underlying mechanism. However, there is evidence that integration is not entirely sequence-autonomous, but depends also on the sequence context, from very closely spaced transmembrane segments to the folding state and properties of neighboring sequences. Topogenesis is even influenced by accessory proteins that appear to act as intramembrane chaperones.

Structural insights into the aggregation mechanism of huntingtin exon 1 protein fragment with different polyQ-lengths.

Huntington disease is a neurodegenerative disorder caused by the expansion of polyglutamine (polyQ) at the N-terminal of the huntingtin exon 1 protein. The detailed structure and the mechanism behind this aggregation remain unclear and it is assumed that the polyQ undergoes a conformational transition to the β-sheet structure when it aggregates. Investigating the misfolding of polyQ facilitates the determination of the molecular mechanism of aggregation and can potentially help in developing a novel approach to inhibit polyQ aggregation. Moreover, the flanking sequences of the polyQ region play a vital role in structural changes and the aggregation mechanism. We performed all-atom molecular dynamics simulations to gain structural insights into the aggregation mechanism using eight different models with glutamine repeat lengths Q27 , Q27 P11 , Q34 , Q35 , Q36 , Q40 , Q50 , and Q50 P11 . In the models without flanking polyPs, we noticed that the transformation of a random coil to β-sheet occurs when the number of Q increases. We also found that the flanking polyPs prevent aggregation by decreasing the probability of forming a β-sheet structure. When polyQ length increases, the 17 N-terminal flanking residues are more likely to adopt a β-sheet conformation from α-helix and coil. From our simulations, we suggest that at least 34 glutamines are required for initiating aggregation and 40 residues length is critical for the aggregation of huntingtin exon 1 protein for disease onset. This study provides structural insights into misfolding and the role of flanking sequences in huntingtin aggregation which will further help in developing therapeutic strategies for Huntington's disease.

Phosphorylation of translation initiation factor eIF2α at Ser51 depends on site- and context-specific information.

Protein kinases phosphorylate specific amino acid residues of substrate proteins and regulate many cellular processes. Specificity for phosphorylation depends on the accessibility of these residues, and more importantly, kinases have preferences for certain residues flanking the phospho-acceptor site. Translation initiation factor 2α [eukaryotic translation initiation factor 2α (eIF2α)] kinase phosphorylates serine51 (Ser51) of eIF2α and downregulates cellular protein synthesis. Structural information on eIF2α reveals that Ser51 is located within a flexible loop, referred to as the Ser51 loop. Recently, we have shown that conformational change of the Ser51 loop increases the accessibility of Ser51 to the kinase active site for phosphorylation. Here, we show that the specificity of Ser51 phosphorylation depends largely on its relative position in the Ser51 loop and minimally on the flanking residues.

Variations in nuclear localization strategies among pol X family enzymes.

Despite the essential roles of pol X family enzymes in DNA repair, information about the structural basis of their nuclear import is limited. Recent studies revealed the unexpected presence of a functional nuclear localization signal (NLS) in DNA polymerase β, indicating the importance of active nuclear targeting, even for enzymes likely to leak into and out of the nucleus. The current studies further explore the active nuclear transport of these enzymes by identifying and structurally characterizing the functional NLS sequences in the three remaining human pol X enzymes: terminal deoxynucleotidyl transferase (TdT), DNA polymerase mu (pol μ) and DNA polymerase lambda (pol λ). NLS identifications are based on Importin α (Impα) binding affinity determined by fluorescence polarization of fluorescein-labeled NLS peptides, X-ray crystallographic analysis of the Impα∆IBB•NLS complexes and fluorescence-based subcellular localization studies. All three polymerases use NLS sequences located near their N-terminus; TdT and pol μ utilize monopartite NLS sequences, while pol λ utilizes a bipartite sequence, unique among the pol X family members. The pol μ NLS has relatively weak measured affinity for Impα, due in part to its proximity to the N-terminus that limits non-specific interactions of flanking residues preceding the NLS. However, this effect is partially mitigated by an N-terminal sequence unsupportive of Met1 removal by methionine aminopeptidase, leading to a 3-fold increase in affinity when the N-terminal methionine is present. Nuclear targeting is unique to each pol X family enzyme with variations dependent on the structure and unique functional role of each polymerase.

Abstract 685: Structural study of the 3'-end G-quadruplex formed in the human PDGFR-β promoter: Insight into a transcriptional inhibitor element

Abstract Aberrant expression of PDGFR-β promotes multiple hallmarks of cancer, and its transcriptional regulation provides an attractive means to inhibit the PDGFR-β signaling pathway. The human PDGFR-β promoter nuclease hypersensitive element (NHE) can form multiple G-quadruplexes. While the most stable G-quadruplex formed in the human PDGFR-β promoter NHE region is the 5'-mid G-quadruplex, the 3'-end sequence that contains a 3'-GGA run forms a less stable G-quadruplex. Interestingly, the G-quadruplex formed in the 3'-end region of the NHE is recently found to be a transcriptional repressor of PDGFR-β, and a small molecule ellipticine, GSA1129, is shown to selectively stabilize the 3'-end G-quadruplex and repress PDGFR-β activity in cancer cell lines and in a preclinical animal model. Therefore, understanding the molecular structure of the 3'-end G-quadruplex is important for small molecule targeting in PDGFR-β gene regulation. Using Nuclear Magnetic Resonance (NMR) spectroscopy, we found that the 3'-end G-quadruplex formed in the extended PDGFR-β NHE 3'-end sequence consists of two novel intramolecular end-insertion G-quadruplexes, one with a 3'-non-adjacent flanking guanine inserted into the 3'-external tetrad (3'-insertion-G4), and another with a 5'-non-adjacent flanking guanine inserted into the 5'-external tetrad (5'-insertion-G4), respectively. The two guanines in the GGA-run move up or down within the G-quadruplex to accommodate the inserted guanine. The two end-insertion G-quadruplexes co-exist in equilibrium under physiological salt conditions. In the 3'-insertion-G4, the 3' non-adjacent guanine G20 is involved in the formation of the 3'-external G-tetrad, with the two guanines from the GGA-run involved in the formation of the 5'-external and middle G-tetrads, thereby inducing the formation of a bulge loop structure from the 3'-flanking strand. In the 5'-insertion-G4, the 5'-non-adjacent flanking guanine G0 is involved in the formation of the 5'-external G-tetrad, with the two guanines from the GGA-run involved in the formation of the middle and 3'-external G-tetrads, thereby inducing the formation of a lateral loop from the 5'-flanking residues on top of the 5'-external tetrad. The loop interactions and capping structures of the two end-insertion G-quadruplexes appear to be different. For the 3'-insertion-G4, only the 5'-flanking bases stabilize the structure. For the 5'-insertion-G4, the flanking residues at both the 5'- and 3'-ends stabilize the structure, and a favorable capping structure appears to be adopted by the 5'-lateral loop. Significantly, the formation of two equilibrating end-insertion G-quadruplexes appears to depend on the GGA-run and the guanine-containing flanking sequences. The unique end-insertion structures and capping conformations may provide a specific platform for small molecule targeting. Citation Format: Buket Onel, Clement Lin, Danzhou Yang. Structural study of the 3'-end G-quadruplex formed in the human PDGFR-β promoter: Insight into a transcriptional inhibitor element [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 685.

Ranking the Contribution of Ankylosing Spondylitis-associated Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) Polymorphisms to Shaping the HLA-B*27 Peptidome

The Endoplasmic reticulum aminopeptidase I (ERAP1) trims peptides to their optimal size for binding to Major Histocompatibility Complex class I proteins. The natural polymorphism of this enzyme is associated with ankylosing spondylitis (AS) in epistasis with the major risk factor for this disease, HLA-B*27, suggesting a direct relationship between AS and HLA-B*27-bound peptides. Three polymorphisms that affect peptide trimming protect from AS: K528R, D575N/R725Q, and Q730E. We characterized and ranked the effects of each mutation, and their various combinations, by quantitative comparisons of the HLA-B*27 peptidomes from cells expressing distinct ERAP1 variants. Five features were examined: peptide length, N-terminal flanking residues, N-terminal residues of the natural ligands, internal sequences and affinity for B*27:05. Polymorphism at residue 528 showed the largest influence, affecting all five features regardless of peptide length. D575N/R725Q showed a much smaller effect. Yet, when co-occurring with K528R, it further decreased ERAP1 activity. Polymorphism at residue 730 showed a significant influence on peptide length, because of distinct effects on trimming of nonamers compared with longer peptides. Accordingly, multiple features were affected by the Q730E mutation in a length-dependent way. The alterations induced in the B*27:05 peptidome by natural ERAP1 variants with different K528R/Q730E combinations reflected separate and additive effects of both mutations. Thus, the influence of ERAP1 on HLA-B*27 is very diverse at the population level, because of the multiplicity and complexity of ERAP1 variants, and to the distinct effects of their co-occurring polymorphisms, leading to significant modulation of disease risk among HLA-B*27-positive individuals.

Structural definition of hSP-D recognition of Salmonella enterica LPS inner core oligosaccharides reveals alternative binding modes for the same LPS.

The crystal structures of a biologically and therapeutically active recombinant homotrimeric fragment of native human SP-D (hSP-D) complexed with the inner core oligosaccharide of the Salmonella enterica sv Minnesota rough strains R5 and R7 (rough mutant chemotypes Rc and Rd1) have been determined. The structures reveal that hSP-D specifically and preferentially targets the LPS inner core via the innermost conserved Hep-Kdo pair with the flexibility for alternative recognition when this preferred epitope is not available for binding. Hep-Kdo binding is achieved through calcium dependent recognition of the heptose dihydroxyethyl side chain coupled with specific interactions between the Kdo and the binding site flanking residues Arg343 and Asp325 with evidence for an extended binding site for LPS inner cores containing multiple Kdo residues. In one subunit of the R5-bound structure this preferred mode of binding is precluded by the crystal lattice and oligosaccharide is bound through the terminal inner core glucose. The structures presented here thus provide unique multiple insights into the recognition and binding of bacterial LPS by hSP-D. Not only is it demonstrated that hSP-D targets the highly conserved LPS proximal inner core Hep-Kdo motif, but also that hSP-D can recognise either terminal or non-terminal sugars and has the flexibility and versatility to adopt alternative strategies for bacterial recognition, utilising alternative LPS epitopes when the preferred inner core Hep-Kdo disaccharide is not available for binding.

Proteomic analysis and prediction of amino acid variations that influence protein posttranslational modifications.

Accumulative studies have indicated that amino acid variations through changing the type of residues of the target sites or key flanking residues could directly or indirectly influence protein posttranslational modifications (PTMs) and bring about a detrimental effect on protein function. Computational mutation analysis can greatly narrow down the efforts on experimental work. To increase the utilization of current computational resources, we first provide an overview of computational prediction of amino acid variations that influence protein PTMs and their functional analysis. We also discuss the challenges that are faced while developing novel in silico approaches in the future. The development of better methods for mutation analysis-related protein PTMs will help to facilitate the development of personalized precision medicine.

Molecular dynamic simulation of mutated β-catenin in solid pseudopapillary neoplasia of the pancreas.

Solid pseudopapillary neoplasia of the pancreas (SPN) is a rare pancreatic neoplasm that frequently harbors mutations in catenin β1 (CTNNB1, encoding β-catenin) as a part of its molecular pathogenesis. Mutations to CTNNB1 reported in SPN usually occur at the serine/threonine phosphorylation sites, including codons 33, 37 and 41, and the flanking residues of codon 33. On analysis of 3 cases of SPN, mutations to CTNNB1 were detected in codon 32 (D32A and D32Y). As this residue, aspartic acid, is not a direct phosphorylation site of the protein, molecular modeling tools were used to predict the influence of these mutations on the protein structure of β-catenin. A total of three MD simulations (wild-type, D32A, and D32Y) were performed to visualize the conformations of β-catenin under in vivo, aqueous-phase conditions at 37°C. In the wild-type protein, the secondary structure of residues P16-H28 remained helical; we therefore hypothesized that the helical structure of this protein fragment (residues M11-G50) was necessary for phosphorylation of S33 phosphorylation. The loss of the secondary structure in P16-H28 was observed in D32A, losing its helical structure and becoming a turn; however, in the D32Y mutant, the helical structure remained. The present demonstrated that structural changes in the mutated β-catenin protein at D32 could potentially explain the mechanism behind its defective phosphorylation in the pathogenesis of SPN.

Specific β-Turns Precede PPIIL Structures Binding to Allele-Specific HLA-DRβ1* PBRs in Fully-Protective Malaria Vaccine Components.

The 3D structural analysis of 62 peptides derived from highly pathogenic Plasmodium falciparum malaria parasite proteins involved in host cell invasion led to finding a striking association between particular β-turn types located in the N-terminal peripheral flanking residue region (preceding the polyproline II left-handed structures fitting into the HLA-DRβ* allele family) and modified immune protection-inducing protein structure induced long-lasting protective immunity. This is the first time association between two different secondary structures associated with a specific immunological function has been described: full, long-lasting protective immunity.

Computational method for distinguishing lysine acetylation, sumoylation, and ubiquitination using the random forest algorithm with a feature selection procedure.

The post-translational modifications (PTMs) on the side chains of conserved lysine (Lys) residues play important roles in myriad cellular processes, such as histone modification, protein degradation, and regulation of DNA damage responses. To date, several computational methods have been developed to identify different PTMs on Lys residues. However, most of these methods focused on identifying one particular PTM regardless of other types of PTMs. In this study, we first conducted a computational investigation of three types of PTMs (acetylation, sumoylation, and ubiquitination) at the same time by analyzing the protein structure and sequence factors surrounding the substrate Lys residues in these types of PTMs. To extract fully the structural and sequence information around the Lys residues, six types of features were used to encode the peptide segments containing the substrates. Next, through a feature selection method, i.e., maximum relevance minimum redundancy (mRMR), two feature lists, i.e., MaxRel feature list and mRMR feature list, were obtained. For the mRMR feature list, it was applied to extract optimal features of the random forest algorithm for distinguishing three types of PTMs. An optimal classification model with overall accuracy of 0.989 was built. For the MaxRel feature list, we investigated the top-ranked features to uncover the site-preference and residue-preference of Lys residues. The results suggested that the disorder structure and the preference of flanking residues were the most important attributes to distinguish the three types of PTMs, which was consistent with the results reported in previous studies.

Conserved Binding Regions Provide the Clue for Peptide-Based Vaccine Development: A Chemical Perspective.

Synthetic peptides have become invaluable biomedical research and medicinal chemistry tools for studying functional roles, i.e., binding or proteolytic activity, naturally-occurring regions’ immunogenicity in proteins and developing therapeutic agents and vaccines. Synthetic peptides can mimic protein sites; their structure and function can be easily modulated by specific amino acid replacement. They have major advantages, i.e., they are cheap, easily-produced and chemically stable, lack infectious and secondary adverse reactions and can induce immune responses via T- and B-cell epitopes. Our group has previously shown that using synthetic peptides and adopting a functional approach has led to identifying Plasmodium falciparum conserved regions binding to host cells. Conserved high activity binding peptides’ (cHABPs) physicochemical, structural and immunological characteristics have been taken into account for properly modifying and converting them into highly immunogenic, protection-inducing peptides (mHABPs) in the experimental Aotus monkey model. This article describes stereo–electron and topochemical characteristics regarding major histocompatibility complex (MHC)-mHABP-T-cell receptor (TCR) complex formation. Some mHABPs in this complex inducing long-lasting, protective immunity have been named immune protection-inducing protein structures (IMPIPS), forming the subunit components in chemically synthesized vaccines. This manuscript summarizes this particular field and adds our recent findings concerning intramolecular interactions (H-bonds or π-interactions) enabling proper IMPIPS structure as well as the peripheral flanking residues (PFR) to stabilize the MHCII-IMPIPS-TCR interaction, aimed at inducing long-lasting, protective immunological memory.

Resolving the Ligand-Binding Specificity in c-MYC G-Quadruplex DNA: Absolute Binding Free Energy Calculations and SPR Experiment.

We report the absolute binding free energy calculation and surface plasmon resonance (SPR) experiment for ligand binding with the c-MYC G-quadruplex DNA. The unimolecular parallel DNA G-quadruplex formed in nuclease hypersensitivity element III1 of the c-MYC gene promoter regulates the c-MYC transcription and is recognized as an emerging drug target for cancer therapy. Quindoline derivatives have been shown to stabilize the G-quadruplex and inhibit the c-MYC expression in cancer cells. NMR revealed two binding sites located at the 5' and 3' termini of the G-quadruplex. Questions about which site is more favored and the basis for the ligand-induced binding site formation remain unresolved. Here, we employ two absolute binding free energy methods, the double decoupling and the potential of mean force methods, to dissect the ligand-binding specificity in the c-MYC G-quadruplex. The calculated absolute binding free energies are in general agreement with the SPR result and suggest that quindoline has a slight preference for the 5' site. The flanking residues around the two sites undergo significant reorganization as the ligand unbinds, which provides evidence for ligand-induced binding pocket formation. The results help interpret experimental data and inform rational design of small molecules targeting the c-MYC G-quadruplex.

Single Amino Acid Substitution in the Vicinity of a Receptor-Binding Domain Changes Protein-Peptide Binding Affinity.

Using a microarray-based assay, we studied how the substitution of amino acids in the immediate vicinity of the receptor-binding domain on a peptide affects its binding to a protein. Replicates of 802 linear peptides consisting of the variants of WTHPQFAT and LQWHPQAGK, GKFPIPLGKQSG, and NGQFQVWIPGAQK, different by one amino acid, were synthesized on a glass slide with a maskless photolithography. Using a microarray-compatible label-free optical sensor, we measured the binding curves of streptavidin with the synthesized peptides and extracted the streptavidin–peptide affinity constants. We found that (a) the substitution of one residue in the HPQ motif reduces the affinity constant Ka from 108 M–1 by at least 3–4 orders of magnitude, with an exception of HPM; (b) substitution of the immediate flanking residue on the Gln side also causes the affinity to decrease by up to 3–4 orders of magnitude, depending on the substituting residue and the second-neighboring flanking residue; (c) substitution of the flanking residues on the His side has no significant effect on the affinity, possibly due to the strong binding of streptavidin to HPQF and HPQAG motifs. We also found that some of amino acid residues located close to the C-terminus (and the solid surface) improve the yield of peptide synthesis on a glass surface and can be exploited in the fabrication of peptide microarrays.

An in silico and in vitro approach to elucidate the impact of residues flanking the cleavage scissile bonds of FVIII.

Coagulation Factor VIII is activated by an ordered limited thrombin proteolysis with different catalytic efficiency at three P1 Arginine residues: Arg759> Arg1708>Arg391, indicating the flanking residues of the latter to be less optimal. This study aimed to investigate, in silico and in vitro, the impact of possessing hypothetically optimized residues at these three catalytic cleavage sites. The structural impact of the residues flanking Arginine cleavage sites was studied by in silico analysis through comparing the cleavage cleft of the native site with a hypothetically optimized sequence at each site. Moreover, recombinant FVIII proteins were prepared by replacing the sequences flanking native thrombin cleavage sites with the proposed cleavage-optimized sequence. FVIII specific activity was determined by assessing the FVIII activity levels in relation to FVIII antigen levels. We further investigated whether thrombin generation could reflect the haemostatic potential of the variants. Our in silico results show the impact of the residues directly in the cleavage bond, and their neighboring residues on the insertion efficiency of the loop into the thrombin cleavage cleft. Moreover, the in vitro analysis shows that the sequences flanking the Arg1708 cleavage site seem to be the most close to optimal residues for achieving the maximal proteolytic activation and profactor activity of FVIII. The residues flanking the scissile bonds of FVIIII affect the cleavage rates and modulate the profactor activation. We were able to provide insights into the mechanisms of the specificity of thrombin for the P1 cleavage sites of FVIII. Thus, the P4-P2´ residues surrounding Arg1708 of FVIII have the highest impact on rates of thrombin proteolysis which contributes to thrombin activation of the profactor and eventually to the thrombin generation potential.

Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character.

After the resolution of the 3D structure of the Cd9-aggregate of the Littorina littorea metallothionein (MT), we report here a detailed analysis of the metal binding capabilities of the wild type MT, LlwtMT, and of two truncated mutants lacking either the N-terminal domain, Lltr2MT, or both the N-terminal domain, plus four extra flanking residues (SSVF), Lltr1MT. The recombinant synthesis and in vitro studies of these three proteins revealed that LlwtMT forms unique M9-LlwtMT complexes with Zn(II) and Cd(II), while yielding a complex mixture of heteronuclear Zn,Cu-LlwtMT species with Cu(I). As expected, the truncated mutants gave rise to unique M6-LltrMT complexes and Zn,Cu-LltrMT mixtures of lower stoichiometry with respect to LlwtMT, with the SSVF fragment having an influence on their metal binding performance. Our results also revealed a major specificity, and therefore a better metal-coordinating performance of the three proteins for Cd(II) than for Zn(II), although the analysis of the Zn(II)/Cd(II) displacement reaction clearly demonstrates a lack of any type of cooperativity in Cd(II) binding. Contrarily, the analysis of their Cu(I) binding abilities revealed that every LlMT domain is prone to build Cu4-aggregates, the whole MT working by modules analogously to, as previously described, certain fungal MTs, like those of C. neoformans and T. mesenterica. It is concluded that the Littorina littorea MT is a Cd-specific protein that (beyond its extended binding capacity through an additional Cd-binding domain) confers to Littorina littorea a particular adaptive advantage in its changeable marine habitat.

Abstract 5225: Novel loop interactions within a parallel-stranded G-quadruplex formed in the human BCL-2 proximal promoter

Abstract The human BCL-2 gene contains a 39-bp GC-rich region upstream of its P1 promoter which has been shown to be critical for BCL-2 transcriptional regulation. Therapeutic inhibition of BCL-2 expression can reduce cell proliferation and the apoptotic threshold of tumorigenic cells. Previously, we have reported that the major G-quadruplex (G4) formed in the Pu39 G-rich sequence adopts a stable, parallel-stranded structure with two 1-nt short loops and a unique 13-nt long central loop. Parallel G-quadruplexes have been found to be prevalent in promoter sequences; the 13-nt loop in BCL-2 Pu39G4 is the longest in this prototype parallel G-quadruplexes identified in human gene promoters. The structure of this G4 with such a long loop is important because it can enable specific small molecule recognition of this BCL-2 G-quadruplex over other G-quadruplexes and provide a potential basis for rational drug design targeting BCL-2 transcription. Thus, the objective of our study is to determine the molecular structure of the BCL-2 Pu39 G-quadruplex and the molecular interactions of this long loop in physiologically relevant K+ solution by Nuclear Magnetic Resonance (NMR) spectroscopy. Our NMR study shows that the BCL-2 Pu39G4 adopts a well-defined parallel-stranded G-quadruplex structure which contains three G-tetrads stabilized by Hoogsteen hydrogen bonding and three double-chain reversal loops containing 1:13:1 bases. The two 1-nt double-chain reversal loops on two edges of the tetrad provide overall stability of the BCL-2 Pu39G4. The central 13-nt double-chain reversal long loop is more dynamic in conformation. Specific interactions of the long loop are observed at both the 5’ and 3’ end with external tetrads as well as the flanking residues, which may also contribute the stable formation of this G4. The middle region of the 13-nt central long loop is highly dynamic and doesn’t show much interaction with the G-quadruplex core. The specific molecular interactions of the unique long central loop are specific to the BCL-2 Pu39G4 sequence/structure and significantly differ from those in other parallel-stranded structures, e.g. c-myc and VEGF. The specific interactions are supported by our mutational analyses. The results are also supported by the DMS footprinting results which show that the guanines involved in the tetrad formation in this major G-quadruplex are well-protected against DMS methylation. Therefore, the knowledge about the molecular structure of this non B-DNA conformation of the BCL-2 promoter region is essential for using the G-quadruplex as a target for anticancer drugs, which could be a novel approach to anti-BCL-2 drug discovery in cancer therapy. Citation Format: Guanhui Wu, Clement Lin, Danzhou Yang. Novel loop interactions within a parallel-stranded G-quadruplex formed in the human BCL-2 proximal promoter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5225. doi:10.1158/1538-7445.AM2017-5225

Refined topology model of the STT3/Stt3 protein subunit of the oligosaccharyltransferase complex

The oligosaccharyltransferase complex, localized in the endoplasmic reticulum (ER) of eukaryotic cells, is responsible for the N-linked glycosylation of numerous protein substrates. The membrane protein STT3 is a highly conserved part of the oligosaccharyltransferase and likely contains the active site of the complex. However, understanding the catalytic determinants of this system has been challenging, in part because of a discrepancy in the structural topology of the bacterial versus eukaryotic proteins and incomplete information about the mechanism of membrane integration. Here, we use a glycosylation mapping approach to investigate these questions. We measured the membrane integration efficiency of the mouse STT3-A and yeast Stt3p transmembrane domains (TMDs) and report a refined topology of the N-terminal half of the mouse STT3-A. Our results show that most of the STT3 TMDs are well inserted into the ER membrane on their own or in the presence of the natural flanking residues. However, for the mouse STT3-A hydrophobic domains 4 and 6 and yeast Stt3p domains 2, 3a, 3c, and 6 we measured reduced insertion efficiency into the ER membrane. Furthermore, we mapped the first half of the STT3-A protein, finding two extra hydrophobic domains between the third and the fourth TMD. This result indicates that the eukaryotic STT3 has 13 transmembrane domains, consistent with the structure of the bacterial homolog of STT3 and setting the stage for future combined efforts to interrogate this fascinating system.

Antimicrobial Activity of Truncated and Polyvalent Peptides Derived from the FKCRRQWQWRMKKGLA Sequence against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923.

Peptides derived from LfcinB were designed and synthesized, and their antibacterial activity was tested against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Specifically, a peptide library was constructed by systemically removing the flanking residues (N or C-terminal) of Lfcin 17–31 (17FKCRRWQWRMKKLGA31), maintaining in all peptides the 20RRWQWR25 sequence that corresponds to the minimal antimicrobial motif. For this research, also included were (i) a peptide containing an Ala instead of Cys ([Ala19]-LfcinB 17–31) and (ii) polyvalent peptides containing the RRWQWR sequence and a non-natural amino acid (aminocaproic acid). We established that the lineal peptides LfcinB 17–25 and LfcinB 17–26 exhibited the greatest activity against E. coli ATCC 25922 and S. aureus ATCC 25923, respectively. On the other hand, polyvalent peptides, a dimer and a tetramer, exhibited the greatest antibacterial activity, indicating that multiple copies of the sequence increase the activity. Our results suggest that the dimeric and tetrameric sequence forms potentiate the antibacterial activity of lineal sequences that have exhibited moderate antibacterial activity.