Identification of a novel rhabdovirus in Spodoptera frugiperda cell lines.

UNLABELLED: The Sf9 cell line, derived from Spodoptera frugiperda, is used as a cell substrate for biological products, and no viruses have been reported in this cell line after extensive testing. We used degenerate PCR assays and massively parallel sequencing (MPS) to identify a novel RNA virus belonging to the order Mononegavirales in Sf9 cells. Sequence analysis of the assembled virus genome showed the presence of five open reading frames (ORFs) corresponding to the genes for the N, P, M, G, and L proteins in other rhabdoviruses and an unknown ORF of 111 amino acids located between the G- and L-protein genes. BLAST searches indicated that the S. frugiperda rhabdovirus (Sf-rhabdovirus) was related in a limited region of the L-protein gene to Taastrup virus, a newly discovered member of the Mononegavirales from a leafhopper (Hemiptera), and also to plant rhabdoviruses, particularly in the genus Cytorhabdovirus. Phylogenetic analysis of sequences in the L-protein gene indicated that Sf-rhabdovirus is a novel virus that branched with Taastrup virus. Rhabdovirus morphology was confirmed by transmission electron microscopy of filtered supernatant samples from Sf9 cells. Infectivity studies indicated potential transient infection by Sf-rhabdovirus in other insect cell lines, but there was no evidence of entry or virus replication in human cell lines. Sf-rhabdovirus sequences were also found in the Sf21 parental cell line of Sf9 cells but not in other insect cell lines, such as BT1-TN-5B1-4 (Tn5; High Five) cells and Schneider's Drosophila line 2 [D.Mel.(2); SL2] cells, indicating a species-specific infection. The results indicate that conventional methods may be complemented by state-of-the-art technologies with extensive bioinformatics analysis for identification of novel viruses. IMPORTANCE: The Spodoptera frugiperda Sf9 cell line is used as a cell substrate for the development and manufacture of biological products. Extensive testing has not previously identified any viruses in this cell line. This paper reports on the identification and characterization of a novel rhabdovirus in Sf9 cells. This was accomplished through the use of next-generation sequencing platforms, de novo assembly tools, and extensive bioinformatics analysis. Rhabdovirus identification was further confirmed by transmission electron microscopy. Infectivity studies showed the lack of replication of Sf-rhabdovirus in human cell lines. The overall study highlights the use of a combinatorial testing approach including conventional methods and new technologies for evaluation of cell lines for unexpected viruses and use of comprehensive bioinformatics strategies for obtaining confident next-generation sequencing results.

Characterization of Sf9 cell clones with differential susceptibilities to Sf-rhabdovirus X+3.7 and Sf-rhabdovirus X- replication.

Our laboratory previously discovered a novel rhabdovirus in the Spodoptera frugiperda Sf9 insect cell line that was designated as Sf-rhabdovirus. Using limiting dilution, this cell line was found to be a mixed population of cells infected by Sf-rhabdovirus variants containing either the full length X accessory gene with a 3.7 kb internal duplication (designated as Sf-rhabdovirus X+3.7) or lacking the duplication and part of the X gene (designated as Sf-rhabdovirus X-), and cells that were negative for Sf-rhabdovirus. In this paper, we found that the Sf-rhabdovirus negative cell clones had sub-populations with different susceptibilities to the replication of Sf-rhabdovirus X+3.7 and X- variants: cell clone Sf9-13F12 was more sensitive to replication by both virus variants compared to Sf9-3003; moreover, Sf9-3003 showed more resistance to X+3.7 replication than to X- replication. RNA-Seq analysis indicated significant differentially expressed genes in the Sf9-13F12 and Sf9-3003 cell clones further supporting that distinct sub-populations of virus-negative cells co-exist in the parent Sf9 cell line.

Long-Read High-Throughput Sequencing (HTS) Revealed That the Sf-Rhabdovirus X+ Genome Contains a 3.7 kb Internal Duplication.

We previously reported a novel rhabdovirus produced from the Spodoptera frugiperda Sf9 cell line, designated as Sf-rhabdovirus X+ since it contained a unique accessory gene X. The Sf-rhabdovirus X+ genome sequence was generated using Sanger sequencing and short-read high-throughput sequencing (HTS). In this study, we have used long-read HTS technologies, PacBio's single-molecule real-time sequencing and Oxford's Nanopore RNA direct sequencing, to analyze the parent Sf9 cell line transcriptome and the virus RNA produced from an X+ cell clone, respectively. A unique 3.7 kb duplication was identified in the L gene between nucleotide position 8523 and 8524, preceded by a GA dinucleotide insertion. This duplication contained a partial G gene, the complete X gene, and a partial L gene, which extended from nucleotide positions 4767-8523 in the X+ virus. Thus, the X+ genome length is 17,361 nucleotides, and we have re-designated the virus as Sf-rhabdovirus X+3.7. The 3.7 kb duplication was found in all Sf9 cell clones producing the X+ variant virus. Furthermore, the Sf-rhabdovirus X+3.7 genome was stable at passage 30, which was the highest passage tested. These results highlight the importance of combining short-read and long-read technologies for accurately sequencing virus genomes using HTS.

Chemical induction of endogenous retrovirus particles from the vero cell line of African green monkeys.

Endogenous retroviral sequences are present in high copy numbers in the genomes of all species and may be expressed as RNAs; however, the majority are defective for virus production. Although virus has been isolated from various Old World monkey and New World monkey species, there has been no report of endogenous retroviruses produced from African green monkey (AGM) tissues or cell lines. We have recently developed a stepwise approach for evaluating the presence of latent viruses by chemical induction (Khan et al., Biologicals 37:196-201, 2009). Based upon this strategy, optimum conditions were determined for investigating the presence of inducible, endogenous retroviruses in the AGM-derived Vero cell line. Low-level reverse transcriptase activity was produced with 5-azacytidine (AzaC) and with 5'-iodo-2'-deoxyuridine (IUdR); none was detected with sodium butyrate. Nucleotide sequence analysis of PCR-amplified fragments from the gag, pol, and env regions of RNAs, prepared from ultracentrifuged pellets of filtered supernatants, indicated that endogenous retrovirus particles related to simian endogenous type D betaretrovirus (SERV) sequences and baboon endogenous virus type C gammaretrovirus (BaEV) sequences were induced by AzaC, whereas SERV sequences were also induced by IUdR. Additionally, sequence heterogeneity was seen in the RNAs of SERV- and BaEV-related particles. Infectivity analysis of drug-treated AGM Vero cells showed no virus replication in cell lines known to be susceptible to type D simian retroviruses (SRVs) and to BaEV. The results indicated that multiple, inducible endogenous retrovirus loci are present in the AGM genome that can encode noninfectious, viruslike particles.

Investigation of xenotropic murine leukemia virus-related virus (XMRV) in human and other cell lines.

Xenotropic murine leukemia virus-related virus (XMRV) was discovered in human prostate tumors and later in some chronic fatigue syndrome (CFS) patients. However, subsequent studies have identified various sources of potential contamination with XMRV and other murine leukemia virus (MLV)-related sequences in test samples. Biological and nucleotide sequence analysis indicates that XMRV is distinct from known xenotropic MLVs and has a broad host range and cell tropism including human cells. Therefore, it is prudent to minimize the risk of human exposure to infection by evaluating XMRV contamination in cell lines handled in laboratory research and particularly those used in the manufacture of biological products. Nested DNA PCR assays were optimized for investigating XMRV gag and env sequences in various cell lines, which included MRC-5, Vero, HEK-293, MDCK, HeLa, and A549, that may be used in the development of some vaccines and other cell lines broadly used in research. The sensitivity of the DNA PCR assays was <10 copies in approximately 1.8 x 10(5) cells equivalent of human DNA. The results indicated the absence of XMRV in the cell lines tested; although in some cases DNA fragments identified as cellular sequences were seen following the first round of PCR amplification with the env primer pair.

Optimization of chemical induction conditions for human herpesvirus 8 (HHV-8) reactivation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) from latently-infected BC-3 cells.

Human herpesvirus 8 (HHV-8) persists as episomal DNA in latently-infected cells and can establish two alternative life cycles, latent or lytic. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is a known inducer of HHV-8 in several human primary effusion lymphoma cell lines and has been widely used for HHV-8 reactivation; however, induction conditions have differed, resulting in varying levels of virus expression. We have used HHV-8 latently-infected BC-3 cells as a model to determine critical parameters for optimizing virus reactivation by TPA. We found that cell growth properties and drug treatment conditions were important for maximum reactivation of HHV-8. Addition of TPA to cells in the early log phase of a sigmoidal growth curve, which was tightly associated with high percentage of the cells in early S phase and with lower histone deacetylase activity in the cells, provided the optimum cell conditions for latent virus to switch to lytic replication. Furthermore, increasing TPA concentration (up to 320 ng per ml) at 48 h exposure time resulted in increased virus production. The results demonstrate the use of a step-wise strategy with chemical induction that may facilitate broad detection of latent DNA viruses and novel virus discovery.

Proposed algorithm to investigate latent and occult viruses in vaccine cell substrates by chemical induction.

The recent urgency to develop new vaccines for emerging and re-emerging diseases, such as pandemic influenza, has necessitated the use of cell substrates not previously used in the manufacture of licensed vaccines. A major safety concern in the use of novel cell substrates is the presence of potential adventitious agents, such as latent and occult viruses, that may not be detected by currently used conventional assays. In cases where the novel cell substrate is known to be tumorigenic, there are additional safety issues related to tumorigenicity of intact cells and oncogenicity of residual cellular DNA. We have developed a strategy for evaluating vaccine cell substrates for the presence of latent/occult viruses, including endogenous retroviruses, latent RNA viruses and oncogenic DNA viruses, by optimizing conditions for chemical induction of viruses and using a combination of broad and specific assays to enable detection of known and novel viruses.

The Spodoptera frugiperda Sf9 cell line is a heterogeneous population of rhabdovirus-infected and virus-negative cells: Isolation and characterization of cell clones containing rhabdovirus X-gene variants and virus-negative cell clones.

The Sf9 cell line is broadly used for manufacturing baculovirus-expressed viral vaccines. We previously reported the presence of a novel, rhabdovirus in the Sf9 cell line, which contained a unique X gene (Sf-rhabdovirus; designated as X+ in this paper). These results were extended by other reports describing an Sf-rhabdovirus variant in Sf9 cells, which lacked 320 nucleotides encompassing the X-gene and adjacent intergenic region (designated as X- in this paper), and the development of an Sf-rhabdovirus negative cell line. Here, we report that the Sf9 cell line is a mixed-cell population, based upon isolation of cell clones with distinct phenotypes: Sf-rhabdovirus-negative, X+, and X-. We also show that Sf-rhabdovirus X+ and X- variants replicate independently in Sf-rhabdovirus-negative cells. These results shed light on the detection of different rhabdovirus variants by different laboratories using Sf9-derived cell clones and confirm that both X+ and X- viruses are infectious in rhabdovirus-negative Sf9 cells.

Whole-Genome Sequence of the Spodoptera frugiperda Sf9 Insect Cell Line.

The draft whole-genome sequence of the Spodoptera frugiperda Sf9 insect cell line was obtained using long-read PacBio sequence technology and Canu assembly. The final assembled genome consisted of 451 Mbp in 4,577 contigs, with 12,716× mean coverage and a G+C content of 36.53%.

Characterization of Ca2+ ATPase residues involved in substrate and cation binding.

The role of amino acid residues involved in substrate and cation binding was investigated in complementary experiments on Fe(2+)-catalyzed oxidation and cleavage, limited digestion with proteinase K, and mutational analysis. Cleavage at Ser346 was produced by Fe(2+) in the presence of substrate (ATP or AMP-PNP) and Ca(2+), and was attributed to Fe(2+) bound to a Mg(2+) site near Ser346 and neighboring Glu696. Ca(2+)- and ATP-dependent oxidation of the Thr441 side chain was also observed and attributed to Fe(2+) substituting for Mg(2+) in the Mg(2+)-ATP complex bound to the N domain. Mutation of Arg560 or Glu439 within the N domain interfered with nucleotide-dependent ATPase resistance to digestion with proteinase K. Furthermore, mutation of Lys352, Lys684, Thr353, Asp703, or Asp707 within the P domain produced similar interference, consistent with a role of these residues in substrate stabilization at the catalytic site. In a third group of experiments, equilibrium isotherms were obtained with Asn796Ala and Glu309Gln mutants, demonstrating non-cooperative binding of one Ca(2+) per ATPase, as opposed to cooperative binding of two Ca(2+) by WT enzyme. No high-affinity binding by Asp800Asn, Glu771Gln, and Thr799Ala mutants was detected. It was also demonstrated that the conformational transitions involved in enzyme activation and interconversion of Ca(2+) binding and phosphorylation energy, are triggered by Ca(2+) binding to site II and stabilization of Glu309 (M4) and N796 (M6).

New technologies and challenges of novel virus detection.

The use of new cell substrates for the development of biologicals, particularly tumorigenic and tumor-derived cell lines, can pose a major regulatory challenge due to safety concerns related to the presence of novel viruses, latent and occult viruses including oncogenic viruses, and endogenous retroviruses, since these may not be detected by the currently recommended conventional assays. This report is a summary of our laboratory's experiences using advanced nucleic acid-based technologies to evaluate a Madin-Darby canine kidney (MDCK) cell line and the insect Sf9 cell line derived from Spodoptera frugiperda, and presents some ongoing efforts to address the challenges of novel virus detection.

Evaluation of the broad-range PCR-electrospray ionization mass spectrometry (PCR/ESI-MS) system and virus microarrays for virus detection.

Advanced nucleic acid-based technologies are powerful research tools for novel virus discovery but need to be standardized for broader applications such as virus detection in biological products and clinical samples. We have used well-characterized retrovirus stocks to evaluate the limit of detection (LOD) for broad-range PCR with electrospray ionization mass spectrometry (PCR/ESI-MS or PLEX-ID), RT-PCR assays, and virus microarrays. The results indicated that in the absence of background cellular nucleic acids, PLEX-ID and RT-PCR had a similar LOD for xenotropic murine retrovirus-related virus (XMRV; 3.12 particles per µL) whereas sensitivity of virus detection was 10-fold greater using virus microarrays. When virus was spiked into a background of cellular nucleic acids, the LOD using PLEX-ID remained the same, whereas virus detection by RT-PCR was 10-fold less sensitive, and no virus could be detected by microarrays. Expected endogenous retrovirus (ERV) sequences were detected in cell lines tested and known species-specific viral sequences were detected in bovine serum and porcine trypsin. A follow-up strategy was developed using PCR amplification, nucleotide sequencing, and bioinformatics to demonstrate that an RD114-like retrovirus sequence that was detected by PLEX-ID in canine cell lines (Madin-Darby canine kidney (MDCK) and Cf2Th canine thymus) was due to defective, endogenous gammaretrovirus-related sequences.

Detection of Latent Retroviruses in Vaccine-related Cell Substrates: Investigation of RT Activity Produced by Chemical Induction of Vero Cells.

CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1-3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA) The detection of known and novel viruses is important for cell substrate and vaccine safety. A major challenge is detection of latent viruses such as endogenous retroviruses and oncogenic DNA viruses. We have evaluated activation of endogenous retroviruses in a Vero cell line using chemical induction and various conventional and emerging methods for virus detection and characterization. In addition, infectivity studies were done to determine whether any induced virus particles were replication competent. This approach may be used for enhancing vaccine safety by assessing the presence of potential chemically-inducible, latent viruses in cell substrates to be used for vaccine manufacture.

Investigations of porcine circovirus type 1 (PCV1) in vaccine-related and other cell lines.

Porcine circovirus type 1 (PCV1) is highly prevalent in swine and was recently reported in some rotavirus vaccines. Since animal-derived raw materials, such as cells, trypsin, and serum, can be a major source of introducing virus contamination in biological products, we have investigated PCV1 in several cell lines obtained from ATCC that have broad use in research, diagnostics, or vaccine development. It is expected that these cell lines have been exposed to bovine and porcine viruses during their establishment and passage history due to the use of serum and trypsin that was not qualified according to current testing guidances or processed using new virus-inactivation methods. This study showed that Vero, MRC-5, and CEFs, which represent cell substrates used in some U.S. licensed vaccines, and other cell lines used in investigational vaccines, such as MDCK, HEK-293, HeLa, and A549, were negative for PCV1 using a nested PCR assay; some were also confirmed negative by infectivity analysis. However, MDBK cells, which are used for some animal vaccines, contained PCV1 sequences, although no virus was isolated. Although the results showed that PCV infection may not have occurred under previous culture conditions, the recent cases of vaccine contamination emphasizes the need for continued efforts to reduce the likelihood of introducing viruses from animal-derived materials used in product manufacture.

Concerted conformational effects of Ca2+ and ATP are required for activation of sequential reactions in the Ca2+ ATPase (SERCA) catalytic cycle.

We relate solution behavior to the crystal structure of the Ca2+ ATPase (SERCA). We find that nucleotide binding occurs with high affinity through interaction of the adenosine moiety with the N domain, even in the absence of Ca2+ and Mg2+, or to the closed conformation stabilized by thapsigargin (TG). Why then is Ca2+ crucial for ATP utilization? The influence of adenosine 5'-(beta,gamma-methylene) triphosphate (AMPPCP), Ca2+, and Mg2+ on proteolytic digestion patterns, interpreted in the light of known crystal structures, indicates that a Ca2+-dependent conformation of the ATPase headpiece is required for a further transition induced by nucleotide binding. This includes opening of the headpiece, which in turn allows inclination of the "A" domain and bending of the "P" domain. Thereby, the phosphate chain of bound ATP acquires an extended configuration allowing the gamma-phosphate to reach Asp351 to form a complex including Mg2+. We demonstrate by Asp351 mutation that this "productive" conformation of the substrate-enzyme complex is unstable because of electrostatic repulsion at the phosphorylation site. However, this conformation is subsequently stabilized by covalent engagement of the -phosphate yielding the phosphoenzyme intermediate. We also demonstrate that the ADP product remains bound with high affinity to the transition state complex but dissociates with lower affinity as the phosphoenzyme undergoes a further conformational change (i.e., E1-P to E2-P transition). Finally, we measured low-affinity ATP binding to stable phosphoenzyme analogues, demonstrating that the E1-P to E2-P transition and the enzyme turnover are accelerated by ATP binding to the phosphoenzyme in exchange for ADP.

Interference with phosphoenzyme isomerization and inhibition of the sarco-endoplasmic reticulum Ca2+ ATPase by 1,3-dibromo-2,4,6-tris(methylisothiouronium) benzene.

ATP hydrolysis and Ca(2+) transport by the sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) are inhibited by 1,3-dibromo-2,4,6-tris(methylisothiouronium) benzene (Br(2)-TITU) in the micromolar range (Berman, M. C., and Karlish, S. J. (2003) Biochemistry 42, 3556-3566). In a study of the mechanism of inhibition, we found that Br(2)-TITU allows the enzyme to bind Ca(2+) and undergo phosphorylation by ATP. The level of ADP-sensitive phosphoenzyme (i.e. E1P-2Ca(2+)) observed in the transient state following addition of ATP is much higher in the presence than in the absence of the inhibitor. Br(2)-TITU does not interfere with enzyme phosphorylation by P(i) in the reverse direction of the cycle (i.e. E2P) and produces only a slight inhibition of its hydrolytic cleavage. The inhibitory effect of Br(2)-TITU on steady state ATPase velocity is attributed to interference with the E1P-2Ca(2+) to E2P-2Ca(2+) transition. In fact, experiments on conformation-dependent protection from proteolytic digestion suggest that, in the presence of Br(2)-TITU, the loops connecting the "A" domain to the ATPase transmembrane region undergo greater fluctuation than expected in the E2 and E2P states. Optimal stability of the gathered headpiece domains is thereby prevented. These effects are opposite to those of thapsigargin, in which the mechanism of inhibition is related to stabilization of a highly compact ATPase conformation and interference with Ca(2+) binding and phosphoenzyme formation. Our experiments with Br(2)-TITU provide the first demonstration of a kinetic limit posed by an inhibitor on the E1P-2Ca(2+) to E2P-2Ca(2+) transition in the wild-type enzyme.

Studies of Ca2+ ATPase (SERCA) inhibition.

The Ca(2+) transport ATPase of intracellular membranes (SERCA) can be inhibited by a series of chemical compounds such as Thapsigargin (TG), 2,5-di(tert-butyl)hydroquinone (DBHQ) and 1,3-dibromo-2,4,6-tris (methyl-isothio-uronium) benzene (Br(2)-TITU). These compounds have specific binding sites in the ATPase protein, and different mechanisms of inhibition. On the other hand, SERCA gene silencing offers a convenient and specific method for suppression of SERCA activity in cells. The physiological and pharmacological implications of SERCA inhibition are discussed.

Functional and structural roles of critical amino acids within the"N", "P", and "A" domains of the Ca2+ ATPase (SERCA) headpiece.

Twenty five amino acids within the "N", "P", and "A" domains of the Ca(2+) ATPase (SERCA1) headpiece were subjected to site directed mutagenesis, taking advantage of a high yield expression system. Functional and conformational effects of mutations were interpreted systematically in the light of the high resolution WT structure, defining direct involvement in catalysis as well as in stabilization of various positions acquired by each domain upon substrate binding and utilization. Amino acids involved in binding of ATP (such as Phe487 and Arg560 in the N domain) or phosphate (such as Asp351, Thr625, Lys684, and Thr353 in the P domain) were characterized with respect to their binding mechanism. Further identified were "positional" roles of several amino acids that stabilize neighboring residues for optimal binding of substrate or Mg(2+), or interface between headpiece domains as they change their relative positions in the course of the catalytic cycle. These include cross-linking of the "N" and "P" domains (e.g., Arg560/Asp627 salt bridge to stabilize domain approximation by ATP binding), and stabilization of the "A", "N", and activated "P" domains in arrangements differing from the ground E2 state and driven by catalytic events. This stabilization is produced through hydrogen bonds at domain interfaces, which vary depending on the intermediate state (e.g., Glu486/T171 in E1P and E2P, as opposed to Glu486/H190 in E2). We demonstrate that specific arrangements of the headpiece domains shown in crystal structures are, in fact, required to trigger displacement of transmembrane segments during the enzyme cycle in solution, allowing long range linkage of catalytic and Ca(2+) binding functions.

Substrate-induced conformational fit and headpiece closure in the Ca2+ATPase (SERCA).

Protection of the Ca2+ATPase (SERCA) from proteinase K digestion has been observed following the addition of Ca2+, Mg2+, and nucleotide and interpreted as a substrate-dependent conformational change (1). The protected digestion site is located on the loop connecting the A domain and the M3 transmembrane helix. We studied by mutational analysis the protective effect of AMP-PCP, an ATP analog that is not utilized for enzyme phosphorylation. We found that the nucleotide protective effect is interfered with by single mutations of Arg-560 and Glu-439 in the N domain and Lys-352, Lys-684, Thr-353, Asp-703, and Asp-707 in the P domain. This is consistent with a transition from the open to the compact configuration of the ATPase headpiece and approximation of the N and P domains by interactions with the nucleotide adenosine and phosphate moieties, respectively. The A domain-M3 loop is consequently involved. Protection by nucleotide substrate increased following the mutations of Asp-351 (the residue undergoing phosphorylation by ATP) and neighboring Asn-706 to Ala, underlying the importance of side chain specificity in positioning the nucleotide terminal phosphate and limiting the stability of the substrate-enzyme complex. Protection is not observed when AMP-PCP is added in the absence of Ca2+ or following mutations (E771Q or N796A) that interfere with Ca2+ binding. Therefore, nucleotide binds to the Ca2+-activated enzyme in the open headpiece conformation and the consequent approximation of the N and P domains occurs while the transmembrane domain is still in the Ca2+-bound conformation. Mg2+ is not required for the protective effect of nucleotide, even though it is specifically required for the subsequent catalytic reactions.

Ca2+ occlusion and gating function of Glu309 in the ADP-fluoroaluminate analog of the Ca2+-ATPase phosphoenzyme intermediate.

In the absence of ATP the sarcoplasmic reticulum ATPase (SERCA) binds two Ca(2+) with high affinity. The two bound Ca(2+) rapidly undergo reverse dissociation upon addition of EGTA, but can be distinguished by isotopic exchange indicating fast exchange at a superficial site (site II), and retardation of exchange at a deeper site (site I) by occupancy of site II. Site II mutations that allow high affinity binding to site I, but only low affinity binding to site II, show that retardation of isotopic exchange requires higher Ca(2+) concentrations with the N796A mutant, and is not observed with the E309Q mutant even at millimolar Ca(2+). Fluoroaluminate forms a complex at the catalytic site yielding stable analogs of the phosphoenzyme intermediate, with properties similar to E2-P or E1-P.Ca(2). Mutational analysis indicates that Asp(351), Lys(352), Thr(353), Asp(703), Asn(706), Asp(707), Thr(625), and Lys(684) participate in stabilization of fluoroaluminate and Mg(2+) at the phosphorylation site. In the presence of fluoroaluminate and Ca(2+), ADP (or AMP-PCP) favors formation of a stable ADP.E1-P.Ca(2) analog. This produces strong occlusion of Ca(2+) bound to both sites (I and II), whereby dissociation occurs very slowly even following addition of EGTA. Occlusion by fluoraluminate and ADP is not observed with the E309Q mutant, suggesting a gating function of Glu(309) at the mouth of a binding cavity with a single path of entry. This phenomenon corresponds to the earliest step of the catalytic cycle following utilization of ATP. Experiments on limited proteolysis reveal that a long range conformational change, involving displacement of headpiece domains and transmembrane helices, plays a mechanistic role.