Conserved Sequence Elements Research Articles

Drosophila Functional Elements Are Embedded in Structurally Constrained Sequences

Modern functional genomics uncovered numerous functional elements in metazoan genomes. Nevertheless, only a small fraction of the typical non-exonic genome contains elements that code for function directly. On the other hand, a much larger fraction of the genome is associated with significant evolutionary constraints, suggesting that much of the non-exonic genome is weakly functional. Here we show that in flies, local (30–70 bp) conserved sequence elements that are associated with multiple regulatory functions serve as focal points to a pattern of punctuated regional increase in G/C nucleotide frequencies. We show that this pattern, which covers a region tenfold larger than the conserved elements themselves, is an evolutionary consequence of a shift in the balance between gain and loss of G/C nucleotides and that it is correlated with nucleosome occupancy across multiple classes of epigenetic state. Evidence for compensatory evolution and analysis of SNP allele frequencies show that the evolutionary regime underlying this balance shift is likely to be non-neutral. These data suggest that current gaps in our understanding of genome function and evolutionary dynamics are explicable by a model of sparse sequence elements directly encoding for function, embedded into structural sequences that help to define the local and global epigenomic context of such functional elements.

Cofactor-dependent specificity of a DEAD-box protein

DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.

RNA PROCESSING FACTOR 5 is required for efficient 5′ cleavage at a processing site conserved in RNAs of three different mitochondrial genes in Arabidopsis thaliana

The 5' ends of many mitochondrial transcripts are generated post-transcriptionally. Recently, we identified three RNA PROCESSING FACTORs required for 5' end maturation of different mitochondrial mRNAs in Arabidopsis thaliana. All of these factors are pentatricopeptide repeat proteins (PPRPs), highly similar to RESTORERs OF FERTILTY (RF), that rescue male fertility in cytoplasmic male-sterile lines from different species. Therefore, we suggested a general role of these RF-like PPRPs in mitochondrial 5' processing. We now identified RNA PROCESSING FACTOR 5, a PPRP not classified as an RF-like protein, required for the efficient 5' maturation of the nad6 and atp9 mRNAs as well as 26S rRNA. The precursor molecules of these RNAs share conserved sequence elements, approximately ranging from positions -50 to +9 relative to mature 5' mRNA termini, suggesting these sequences to be at least part of the cis elements required for processing. The knockout of RPF5 has only a moderate influence on 5' processing of atp9 mRNA, whereas the generation of the mature nad6 mRNA and 26S rRNA is almost completely abolished in the mutant. The latter leads to a 50% decrease of total 26S rRNA species, resulting in an imbalance between the large rRNA and 18S rRNA. Despite these severe changes in RNA levels and in the proportion between the 26S and 18S rRNAs, mitochondrial protein levels appear to be unaltered in the mutant, whereas seed germination capacity is markedly reduced.

Eukaryotic TPP riboswitch regulation of alternative splicing involving long-distance base pairing

Thiamin pyrophosphate (TPP) riboswitches are found in organisms from all three domains of life. Examples in bacteria commonly repress gene expression by terminating transcription or by blocking ribosome binding, whereas most eukaryotic TPP riboswitches are predicted to regulate gene expression by modulating RNA splicing. Given the widespread distribution of eukaryotic TPP riboswitches and the diversity of their locations in precursor messenger RNAs (pre-mRNAs), we sought to examine the mechanism of alternative splicing regulation by a fungal TPP riboswitch from Neurospora crassa, which is mostly located in a large intron separating protein-coding exons. Our data reveal that this riboswitch uses a long-distance (∼530-nt separation) base-pairing interaction to regulate alternative splicing. Specifically, a portion of the TPP-binding aptamer can form a base-paired structure with a conserved sequence element (α) located near a 5′ splice site, which greatly increases use of this 5′ splice site and promotes gene expression. Comparative sequence analyses indicate that many fungal species carry a TPP riboswitch with similar intron architecture, and therefore the homologous genes in these fungi are likely to use the same mechanism. Our findings expand the scope of genetic control mechanisms relying on long-range RNA interactions to include riboswitches.

HnRNPH1/H2, U1 snRNP, and U11 snRNP cooperate to regulate the stability of the U11-48K pre-mRNA

Alternative splicing (AS) is a major contributor to proteome diversity, but it also regulates gene expression by introducing premature termination codons (PTCs) that destabilize transcripts, typically via the nonsense-mediated decay (NMD) pathway. Such AS events often take place within long, conserved sequence elements, particularly in genes encoding various RNA binding proteins. AS-NMD is often activated by the protein encoded by the same gene, leading to a self-regulating feedback loop that maintains constant protein levels. However, cross-regulation between different RNA binding proteins is also common, giving rise to finely tuned regulatory networks. Recently, we described a feedback mechanism regulating two protein components of the U12-dependent spliceosome (U11-48K and U11/U12-65K) through a highly conserved sequence element. These elements contain a U11 snRNP-binding splicing enhancer (USSE), which, through the U11 snRNP, activates an upstream U2-type 3'ss, resulting in the degradation of the U11-48K mRNA by AS-NMD. Through phylogenetic analysis, we now identify a G-rich sequence element that is conserved in fishes as well as mammals. We show that this element binds hnRNPF/H proteins in vitro. Knockdown of hnRNPH1/H2 or mutations in the G-run both lead to enhanced activation of the 3'ss in vivo, suggesting that hnRNPH1/H2 proteins counteract the 3'ss activation. Furthermore, we provide evidence that U1 binding immediately downstream from the G-run similarly counteracts the U11-mediated activation of the alternative 3'ss. Thus, our results elucidate the mechanism in which snRNPs from both spliceosomes together with hnRNPH1/H2 proteins regulate the recognition and activation of the highly conserved alternative splice sites within the U11-48K pre-mRNA.

AtMYB44 regulates WRKY70 expression and modulates antagonistic interaction between salicylic acid and jasmonic acid signaling

The role of AtMYB44, an R2R3 MYB transcription factor, in signaling mediated by jasmonic acid (JA) and salicylic acid (SA) is examined. AtMYB44 is induced by JA through CORONATINE INSENSITIVE1 (COI1). AtMYB44 over-expression down-regulated defense responses against the necrotrophic pathogen Alternaria brassicicola, but up-regulated WRKY70 and PR genes, leading to enhanced resistance to the biotrophic pathogen Pseudomonas syringae pv. tomato DC3000. The knockout mutant atmyb44 shows opposite effects. Induction of WRKY70 by SA is reduced in atmyb44 and npr1-1 mutants, and is totally abolished in atmyb44 npr1-1 double mutants, showing that WRKY70 is regulated independently through both NPR1 and AtMYB44. AtMYB44 over-expression does not change SA content, but AtMYB44 over-expression phenotypes, such as retarded growth, up-regulated PR1 and down-regulated PDF1.2 are reversed by SA depletion. The wrky70 mutation suppressed AtMYB44 over-expression phenotypes, including up-regulation of PR1 expression and down-regulation of PDF1.2 expression. β-estradiol-induced expression of AtMYB44 led to WRKY70 activation and thus PR1 activation. AtMYB44 binds to the WRKY70 promoter region, indicating that AtMYB44 acts as a transcriptional activator of WRKY70 by directly binding to a conserved sequence element in the WRKY70 promoter. These results demonstrate that AtMYB44 modulates antagonistic interaction by activating SA-mediated defenses and repressing JA-mediated defenses through direct control of WRKY70.

Bioinformatic identification of novel elements potentially involved in messenger RNA fate control during spermatogenesis.

In eukaryotic cells, 3' untranslated regions (3' UTRs) of mRNA transcripts contain conserved sequence elements (motifs), which, once bound by RNA-binding proteins, can affect mRNA stability and translational efficacy. Despite abundant sequences contained within the 3' UTRs, only a limited number of motifs are known to interact with RNA-binding proteins and have a role in mRNA fate control. Spermatogenesis represents an excellent in vivo model for studying posttranscriptional regulation of gene expression because numerous mRNAs are transcribed in late pachytene spermatocytes and/or round spermatids, but their translation will not occur until many hours or even days later, when they have developed into elongated spermatids, in which transcription has long been shut off because of the increasingly condensed chromatin. Translationally suppressed mRNAs are sequestered and confined to ribonuclear protein particles, and their loading onto the ribosomes marks their translation. By bioinformatic sequence analyses of the 3' UTRs of translationally suppressed mRNAs during spermatogenesis, we identified numerous novel sequence elements overrepresented in the transcripts subject to posttranscriptional regulation than in the unregulated transcripts. These include AU(U/A)(U/A)UGAGU and (A/U)AUUA(U/C/G) for genes translationally upregulated in early spermiogenesis, and (G/A)GUACG(U/C/A)(A/U)(A/U) and UGUAGC for genes translationally upregulated in late spermiogenesis. The bioinformatic approach reported in this study can be adapted for rapid discovery of novel regulatory elements involved in mRNA fate control in a wide range of tissues or organs.

Dephosphorylation of HuR Protein during Alphavirus Infection Is Associated with HuR Relocalization to the Cytoplasm

We have demonstrated previously that the cellular HuR protein binds U-rich elements in the 3' untranslated region (UTR) of Sindbis virus RNA and relocalizes from the nucleus to the cytoplasm upon Sindbis virus infection in 293T cells. In this study, we show that two alphaviruses, Ross River virus and Chikungunya virus, lack the conserved high-affinity U-rich HuR binding element in their 3' UTRs but still maintain the ability to interact with HuR with nanomolar affinities through alternative binding elements. The relocalization of HuR protein occurs during Sindbis infection of multiple mammalian cell types as well as during infections with three other alphaviruses. Interestingly, the relocalization of HuR is not a general cellular reaction to viral infection, as HuR protein remained largely nuclear during infections with dengue and measles virus. Relocalization of HuR in a Sindbis infection required viral gene expression, was independent of the presence of a high-affinity U-rich HuR binding site in the 3' UTR of the virus, and was associated with an alteration in the phosphorylation state of HuR. Sindbis virus-induced HuR relocalization was mechanistically distinct from the movement of HuR observed during a cellular stress response, as there was no accumulation of caspase-mediated HuR cleavage products. Collectively, these data indicate that virus-induced HuR relocalization to the cytoplasm is specific to alphavirus infections and is associated with distinct posttranslational modifications of this RNA-binding protein.

The chromatin landscape of the casein gene locus

For several decades, the regulation of casein gene expression by the lactogenic hormones, prolactin and glucocorticoids, has provided an excellent model system in which to study how steroid and peptide hormones regulate gene expression. Early studies of casein gene regulation defined conserved sequence elements in the 5' flanking region of these genes, including one of which was identified as a γ-interferon activation sequence (GAS). Although this site was thought to interact with a mammary gland-specific factor, purification and cloning of this factor by Bernd Groner and his colleagues revealed it was instead a new member of the signal transducers and activators of transcription family, Stat5, which was expressed in many tissues. The exquisite tissue-specific expression of the casein genes was subsequently shown to depend not on a single transcription factor but on composite response elements that interacted with a number of ubiquitous transcription factors in response to the combinatorial effects of peptide and steroid hormone signaling. More recent studies have defined cooperative effects of prolactin and glucocorticoids as well as antagonistic effects of progesterone on the chromatin structure of both the casein gene proximal promoter region as well as a distal enhancer. Local chromatin modifications as well as long-range interactions facilitated by DNA looping are required for the hormonal regulation of β-casein gene expression. The casein genes are part of a large gene cluster, and the chromatin landscape of the entire cluster is regulated in a tissue-specific and developmental manner. Finally, newly discovered large non coding RNAs, such as the pregnancy-induced non coding RNA (PINC) may play an important role in the epigenetic regulation of mammary gland differentiation.

Transient Tertiary Contact Formation in the CGRP Neuropeptide Revealed by Nanosecond Laser Spectroscopy

Calcitonin gene-related peptide (CGRP) is an intrinsically disordered, 37 residue neuropeptide that acts as a potent vasodilator, which is of considerable interest in migrane research. It is a member of the calcitonin peptide (Ct) family, together with amylin, calcitonin and adrenomedullin. These are genetically and structurally related intrinsically disordered hormone peptides that are able to bind to each other’s receptors, though with varying degrees of affinity. They contain highly conserved sequence elements that have been experimentally shown to affect the secondary structural preferences of these peptides. The effect of such conserved elements on tertiary structure has not been experimentally explored to the same extent. Detecting tertiary structural properties of IDPs is considerably more challenging due to fast reconfigurations of the backbone over a wide range of possible conformations. High resolution time-resolved techniques are needed.We use a nanosecond-resolved spectroscopic technique based on tryptophan triplet quenching by cystine to detect tertiary contact formation in CGRP under varying solvent and temperature conditions. Using this technique, Vaiana et al.1 have previously shown that conserved structural elements of amylin induce compact states characterized by short end-to-end distances, and that compaction is not driven by hydrophobic side-chains. Using triplet quenching we explore the effect of these conserved elements on the conformation and dynamics of CGRP, a peptide with higher mean hydrophobicity and net charge per residue than amylin. We discuss our findings in relation to secondary structural preferences of these peptides and discuss their possible functional role.Footnote1 Vaiana S.M. et al. Biophys. J. 97 2009.

Identification of CTCF as a master regulator of the clustered protocadherin genes

The brain is a large and complex network of neurons. Specific neuronal connectivity is thought to be based on the combinatorial expression of the 52 protocadherins (Pcdh) membrane adhesion proteins, whereby each neuron expresses only a specific subset. Pcdh genes are arranged in tandem, in a cluster of three families: Pcdhα, Pcdhβ and Pcdhγ. The expression of each Pcdh gene is regulated by a promoter that has a regulatory conserved sequence element (CSE), common to all 52 genes. The mechanism and factors controlling individual Pcdh gene expression are currently unknown. Here we show that the promoter of each Pcdh gene contains a gene-specific conserved control region, termed specific sequence element (SSE), located adjacent and upstream to the CSE and activates transcription together with the CSE. We purified the complex that specifically binds the SSE–CSE region and identified the CCTC binding-factor (CTCF) as a key molecule that binds and activates Pcdh promoters. Our findings point to CTCF as a factor essential for Pcdh expression and probably governing neuronal connectivity.

Genotyping and antifungal susceptibility of Candida albicans clinical isolates from Yangzhou region in China

In this report, 500 clinical isolates of Candida albicans originated from patients’ body sites (vaginal, rectum, tongue and pharynx) and samples (stool, urine) in Yangzhou-located region were selected, screened and characterized. C. albicans DNA fragments spanning a potential transposable group I intron-containing region in 25S r DNA were amplified by polymerase chain reaction (PCR), cloned, sequenced and analyzed. Based on the DNA band, size and sequence data, they could be classified into three genotypes: genotype A (365 strains), genotype B (83 strains) and genotype C (52 strains). Sequence alignment analysis indicated that genotype B contained the conserved sequence element P, Q, R and S of group I intron, genotype C only harbored sequence element P and Q, while genotype A lacked these characteristic element sequences. 80 isolates covering the three genotypes were further selected to perform the in vitro antibiotics susceptibility test. The results indicated that strains of genotype A were much less susceptible to flucytosine than either strains of genotype B or genotype C. But no regularity was observed between the susceptibility of C. albicans isolates to azoles, and to amphotericin B. To our knowledge, this is the first report about the prevalence and antibiotics susceptibility of Chinese-basedC. albicans. Key words: Candida albicans, polymerase chain reaction (PCR), genotyping, antifungal susceptibility.

Identification of the Cluster Control Region for the Protocadherin-β Genes Located beyond the Protocadherin-γ Cluster

The clustered protocadherins (Pcdhs), Pcdh-α, -β, and -γ, are transmembrane proteins constituting a subgroup of the cadherin superfamily. Each Pcdh cluster is arranged in tandem on the same chromosome. Each of the three Pcdh clusters shows stochastic and combinatorial expression in individual neurons, thus generating a hugely diverse set of possible cell surface molecules. Therefore, the clustered Pcdhs are candidates for determining neuronal molecular diversity. Here, we showed that the targeted deletion of DNase I hypersensitive (HS) site HS5-1, previously identified as a Pcdh-α regulatory element in vitro, affects especially the expression of specific Pcdh-α isoforms in vivo. We also identified a Pcdh-β cluster control region (CCR) containing six HS sites (HS16, 17, 17', 18, 19, and 20) downstream of the Pcdh-γ cluster. This CCR comprehensively activates the expression of the Pcdh-β gene cluster in cis, and its deletion dramatically decreases their expression levels. Deleting the CCR nonuniformly down-regulates some Pcdh-γ isoforms and does not affect Pcdh-α expression. Thus, the CCR effect extends beyond the 320-kb region containing the Pcdh-γ cluster to activate the upstream Pcdh-β genes. Thus, we concluded that the CCR is a highly specific regulatory unit for Pcdh-β expression on the clustered Pcdh genomic locus. These findings suggest that each Pcdh cluster is controlled by distinct regulatory elements that activate their expression and that the stochastic gene regulation of the clustered Pcdhs is controlled by the complex chromatin architecture of the clustered Pcdh locus.

The mitochondrial genome of Apodemus peninsulae (Rodentia, Muridae)

The complete mitochondrial (mt) genome of the Korean field mouse Apodemus peninsulae was sequenced and found to be 16,266 bp in length. The mt protein-coding genes of A. peninsulae had ATG, GTG, ATC, and ATA as initiation codons and TAA, TAG, TA, and T as termination codons. Two forms each of trnL and trnS and the three tRNA clusters, IQM, WANCY, and HSL were identified, as in the typical Rodentia mt genome. Among tRNAs, abnormal cloverleaf structure of trnS(AGY) was identified in DHU arm. The l-strand replication origin has the potential to form a stable stem–loop structure and control region has several conserved sequence elements.

In silico Analysis of 3'-End-Processing Signals in Aspergillus oryzae Using Expressed Sequence Tags and Genomic Sequencing Data

To investigate 3′-end-processing signals in Aspergillus oryzae, we created a nucleotide sequence data set of the 3′-untranslated region (3′ UTR) plus 100 nucleotides (nt) sequence downstream of the poly(A) site using A. oryzae expressed sequence tags and genomic sequencing data. This data set comprised 1065 sequences derived from 1042 unique genes. The average 3′ UTR length in A. oryzae was 241 nt, which is greater than that in yeast but similar to that in plants. The 3′ UTR and 100 nt sequence downstream of the poly(A) site is notably U-rich, while the region located 15–30 nt upstream of the poly(A) site is markedly A-rich. The most frequently found hexanucleotide in this A-rich region is AAUGAA, although this sequence accounts for only 6% of all transcripts. These data suggested that A. oryzae has no highly conserved sequence element equivalent to AAUAAA, a mammalian polyadenylation signal. We identified that putative 3′-end-processing signals in A. oryzae, while less well conserved than those in mammals, comprised four sequence elements: the furthest upstream U-rich element, A-rich sequence, cleavage site, and downstream U-rich element flanking the cleavage site. Although these putative 3′-end-processing signals are similar to those in yeast and plants, some notable differences exist between them.

Expansion of the Ago gene family in the teleost clade

AGO proteins are universal effectors of eukaryotic small RNA-directed regulatory pathways. In this study, we used a comparative genomics approach to explore the AGO sub-family in the teleost clade. We identified five Ago homologues in teleost genomes, one more than encoded in other vertebrate clades. The additional teleost homologue was preserved most likely due to the differential retention of regulatory elements following the fish-specific genome duplication event that occurred approximately 350 million years ago. Analysis of all five Ago genomic loci in teleosts revealed that orthologues contain specific, conserved sequence elements in non-coding regions indicating that the teleost Ago paralogues are differentially regulated. This was supported by qRT-PCR analysis that showed differential expression of the zebrafish homologues across development and between adult tissues indicating stage and tissue-specific function of individual AGO proteins. Multiple sequence alignments showed not only that all teleost homologues possess critical residues for AGO function, but also that teleost homologues contain multiple orthologue-specific features, indicative of structural diversification. Notably, these are retained throughout the vertebrate lineage arguing these may be important for orthologue-specific functions.

Identification of an NTPase motif in classical swine fever virus NS4B protein

Classical swine fever (CSF) is a highly contagious and often fatal disease of swine caused by CSF virus (CSFV), a positive-sense single-stranded RNA virus within the Pestivirus genus of the Flaviviridae family. Here, we have identified conserved sequence elements observed in nucleotide-binding motifs (NBM) that hydrolyze NTPs within the CSFV non-structural (NS) protein NS4B. Expressed NS4B protein hydrolyzes both ATP and GTP. Substitutions of critical residues within the identified NS4B NBM Walker A and B motifs significantly impair the ATPase and GTPase activities of expressed proteins. Similar mutations introduced into the genetic backbone of a full-length cDNA copy of CSFV strain Brescia rendered no infectious viruses or viruses with impaired replication capabilities, suggesting that this NTPase activity is critical for the CSFV cycle. Recovered mutant viruses retained a virulent phenotype, as parental strain Brescia, in infected swine. These results have important implications for developing novel antiviral strategies against CSFV infection.

Identification and Characterization of a FYVE Domain from the Early Diverging Eukaryote Giardia lamblia

The morphology of the endomembrane system of Giardia lamblia appears to be significantly different from higher eukaryotes. Therefore, the molecular mechanisms controlling vesicular trafficking are also likely to be altered. Since FYVE domain is a known regulator of endosomal trafficking, the authors used BLAST search to identify FYVE domain(s) in G. lamblia. A 990 amino acid long putative FYVE domain-containing ORF was identified, which contains all the conserved sequence elements in the ligand binding pocket. Phylogenetic analysis reveals that this domain is significantly diverged. The authors have shown that the corresponding gene is expressed in G. lamblia trophozoites and cysts. In spite of this phylogenetic divergence, in vitro biochemical assay indicates that this domain preferentially binds to phosphatidylinositol 3-phosphate {PtdIns(3)P}and in vivo expression of the GFP-tagged G. lamblia FYVE domain in S. cerevisiae, displays its selective localization to PtdIns(3)P-enriched endosomes. This is the first study to characterize a PtdIns(3)P effector protein in this early-diverged eukaryote.

Competing RNA secondary structures are required for mutually exclusive splicing of the Dscam exon 6 cluster

Alternative splicing of eukaryotic pre-mRNAs is an important mechanism for generating proteome diversity and regulating gene expression. The Drosophila melanogaster Down Syndrome Cell Adhesion Molecule (Dscam) gene is an extreme example of mutually exclusive splicing. Dscam contains 95 alternatively spliced exons that potentially encode 38,016 distinct mRNA and protein isoforms. We previously identified two sets of conserved sequence elements, the docking site and selector sequences in the Dscam exon 6 cluster, which contains 48 mutually exclusive exons. These elements were proposed to engage in competing RNA secondary structures required for mutually exclusive splicing, though this model has not yet been experimentally tested. Here we describe a new system that allowed us to demonstrate that the docking site and selector sequences are indeed required for exon 6 mutually exclusive splicing and that the strength of these RNA structures determines the frequency of exon 6 inclusion. We also show that the function of the docking site has been conserved for ~500 million years of evolution. This work demonstrates that conserved intronic sequences play a functional role in mutually exclusive splicing of the Dscam exon 6 cluster.

All-Atom Models of the Membrane-Spanning Domain of HIV-1 gp41 from Metadynamics

The 27-residue membrane-spanning domain (MSD) of the HIV-1 glycoprotein gp41 bears conserved sequence elements crucial to the biological function of the virus, in particular a conserved GXXXG motif and a midspan arginine. However, structure-based explanations for the roles of these and other MSD features remain unclear. Using molecular dynamics and metadynamics calculations of an all-atom, explicit solvent, and membrane-anchored model, we study the conformational variability of the HIV-1 gp41 MSD. We find that the MSD peptide assumes a stable tilted α-helical conformation in the membrane. However, when the side chain of the midspan Arg 694 “snorkels” to the outer leaflet of the viral membrane, the MSD assumes a metastable conformation where the highly-conserved N-terminal core (between Lys 681 and Arg 694 and containing the GXXXG motif) unfolds. In contrast, when the Arg 694 side chain snorkels to the inner leaflet, the MSD peptide assumes a metastable conformation consistent with experimental observations where the peptide kinks at Phe 697 to facilitate Arg 694 snorkeling. Both of these models suggest specific ways that gp41 may destabilize viral membrane, priming the virus for fusion with a target cell.