Ocular biodistribution of cysteamine delivered by a sustained release microsphere/thermoresponsive gel eyedrop.

The objective of the investigation was to determine the ocular biodistribution of cysteamine, a reducing agent used for treatment of cystine crystals in cystinosis, following topical administration of a sustained release formulation and traditional eyedrop formulation. To the right eye only, rabbits received a 50 µL drop of 0.44% cysteamine eyedrops at one drop per waking hour for 2, 6, 12, and 24 h. A second group received one 100 µL drop of a sustained release formulation containing encapsulated cysteamine microspheres suspended in a thermoresponsive gel. Upon serial sacrifice, ocular tissues from both eyes and plasma were obtained and quantified for cysteamine using LC-MS/MS. Cysteamine was detected in the cornea, aqueous humor and vitreous humor. Systemic plasma concentrations of cysteamine from treatment groups were below the limit of detection. As expected, 0.44% cysteamine eyedrops when administered hourly maintained drug concentrations within the cornea at a magnitude 5 times higher than a single dose of the sustained release formulation over 12 h. The sustained release formulation maintained cysteamine presentation across 12 h from a single drop. These studies demonstrate distribution of cysteamine to the eye following topical administration, including high drug uptake to the cornea and low systemic distribution.

Changes in Expression of DNA-Methyltransferase and Cannabinoid Receptor mRNAs in Blood Lymphocytes After Acute Cannabis Smoking.

Background: Cannabis use is a component risk factor for the manifestation of schizophrenia. The biological effects of cannabis include effects on epigenetic systems, immunological parameters, in addition to changes in cannabinoid receptors 1 and 2, that may be associated with this risk. However, there has been limited study of the effects of smoked cannabis on these biological effects in human peripheral blood cells. We analyzed the effects of two concentrations of tetrahydrocannabinol (THC) vs. placebo in lymphocytes of a subset of participants who enrolled in a double-blind study of the effects of cannabis on driving performance (outcome not the focus of this study). Methods: Twenty four participants who regularly use cannabis participated in an experiment in which they smoked cannabis cigarettes (5.9 or 13.4% THC) or placebo (0.02%) ad libitum. Blood samples were drawn at baseline and several times after smoking. Lymphocytes were separated and stored at -80°C for further analysis. Samples were analyzed for mRNA content for cannabinoid receptors 1 (CB1) and 2 (CB2), methylation and demethylating enzymes (DNMT, TET), glucocorticoid receptor (NRC3) and immunological markers (IL1B, TNFα) by qPCR using TaqMan probes. The results were correlated with THC whole blood levels during the course of the day, as well as THCCOOH baseline levels. Statistical analyses used analysis of variance and covariance and t-tests, or non-parametric equivalents for those values which were not normally distributed. Results: There were no differences in background baseline characteristics of the participants except that the higher concentration THC group was older than the low concentration and placebo groups, and the low concentration THC group had higher baseline CB2 mRNA levels. Both the 5.9 and 13.4% THC groups showed increased THC blood levels that then decreased toward baseline within the first hour. However, there were no significant differences between THC blood levels between the 5.9 and 13.4% groups at any time point. At the 4-h time point after drug administration the 13.4% THC group had higher CB2 (P = 0.021) and DNMT3A (P = 0.027) mRNA levels than the placebo group. DNMT1 mRNA levels showed a trend in the same direction (P = 0.056). The higher 13.4% THC group had significantly increased CB2 mRNA levels than the 5.9% concentration group at several post drug administration time points and showed trends for difference in effects for between 5.9 and 13.4% THC groups for other mRNAs. TET3 mRNA levels were higher in the 13.4% THC group at 55 min post-cannabis ingestion. When the high and lower concentration THC groups were combined, none of the differences in mRNA levels from placebo remained statistically significant. Changes in THC blood levels were not related to changes in mRNA levels. Conclusion: Over the time course of this study, CB2 mRNA increased in blood lymphocytes in the high concentration THC group but were not accompanied by changes in immunological markers. The changes in DNMT and TET mRNAs suggest potential epigenetic effects of THC in human lymphocytes. Increases in DNMT methylating enzymes have been linked to some of the pathophysiological processes in schizophrenia and, therefore, should be further explored in a larger sample population, as one of the potential mechanisms linking cannabis use as a trigger for schizophrenia in vulnerable individuals. Since the two THC groups did not differ in post-smoking blood THC concentrations, the relationship between lymphocytic changes and the THC content of the cigarettes remains to be determined.

Cardiomyocytes disrupt pyrimidine biosynthesis in nonmyocytes to regulate heart repair.

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.

Deficiency of the sedoheptulose kinase (Shpk) does not alter the ability of hematopoietic stem cells to rescue cystinosis in the mouse model.

Cystinosis is an autosomal recessive lysosomal storage disorder caused by mutations in the CTNS gene encoding the lysosomal cystine transporter, cystinosin, and leading to multi-organ degeneration including kidney failure. A clinical trial for cystinosis is ongoing to test the safety and efficacy of transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) ex vivo gene-modified to introduce functional CTNS cDNA. Preclinical studies in Ctns-/- mice previously showed that a single HSPC transplantation led to significant tissue cystine decrease and long-term tissue preservation. The main mechanism of action involves the differentiation of the transplanted HSPCs into macrophages within tissues and transfer of cystinosin-bearing lysosomes to the diseased cells via tunneling nanotubes. However, a major concern was that the most common cystinosis-causing mutation in humans is a 57-kb deletion that eliminates not only CTNS but also the adjacent sedopheptulose kinase SHPK/CARKL gene encoding a metabolic enzyme that influences macrophage polarization. Here, we investigated if absence of Shpk could negatively impact the efficiency of transplanted HSPCs to differentiate into macrophages within tissues and then to prevent cystinosis rescue. We generated Shpk knockout mouse models and detected a phenotype consisting of perturbations in the pentose phosphate pathway (PPP), the metabolic shunt regulated by SHPK. Shpk-/- mice also recapitulated the urinary excretion of sedoheptulose and erythritol found in cystinosis patients homozygous for the 57-kb deletion. Transplantation of Shpk-/--HSPCs into Ctns-/- mice resulted in significant reduction in tissue cystine load and restoration of Ctns expression, as well as improved kidney architecture comparable to WT-HSPC recipients. Altogether, these data demonstrate that absence of SHPK does not alter the ability of HSPCs to rescue cystinosis, and then patients homozygous for the 57-kb deletion should benefit from ex vivo gene therapy and can be enrolled in the ongoing clinical trial. However, because of the limits inherent to animal models, outcomes of this patient population will be carefully compared to the other enrolled subjects.

An endogenous peptide marker differentiates SOD1 stability and facilitates pharmacodynamic monitoring in SOD1 amyotrophic lateral sclerosis.

The discovery of novel biomarkers has emerged as a critical need for therapeutic development in amyotrophic lateral sclerosis (ALS). For some subsets of ALS, such as the genetic superoxide dismutase 1 (SOD1) form, exciting new treatment strategies, such as antisense oligonucleotide-mediated (ASO-mediated) SOD1 silencing, are being tested in clinical trials, so the identification of pharmacodynamic biomarkers for therapeutic monitoring is essential. We identify increased levels of a 7-amino acid endogenous peptide of SOD1 in cerebrospinal fluid (CSF) of human SOD1 mutation carriers but not in other neurological cases or nondiseased controls. Levels of peptide elevation vary based on the specific SOD1 mutation (ranging from 1.1-fold greater than control in D90A to nearly 30-fold greater in V148G) and correlate with previously published measurements of SOD1 stability. Using a mass spectrometry-based method (liquid chromatography-mass spectrometry), we quantified peptides in both extracellular samples (CSF) and intracellular samples (spinal cord from rat) to demonstrate that the peptide distinguishes mutation-specific differences in intracellular SOD1 degradation. Furthermore, 80% and 63% reductions of the peptide were measured in SOD1G93A and SOD1H46R rat CSF samples, respectively, following treatment with ASO, with an improved correlation to mRNA levels in spinal cords compared with the ELISA measuring intact SOD1 protein. These data demonstrate the potential of this peptide as a pharmacodynamic biomarker.

Promises and pitfalls of untargeted metabolomics.

Metabolomics is one of the newer omics fields, and has enabled researchers to complement genomic and protein level analysis of disease with both semi-quantitative and quantitative metabolite levels, which are the chemical mediators that constitute a given phenotype. Over more than a decade, methodologies have advanced for both targeted (quantification of specific analytes) as well as untargeted metabolomics (biomarker discovery and global metabolite profiling). Untargeted metabolomics is especially useful when there is no a priori metabolic hypothesis. Liquid chromatography coupled to mass spectrometry (LC-MS) has been the preferred choice for untargeted metabolomics, given the versatility in metabolite coverage and sensitivity of these instruments. Resolving and profiling many hundreds to thousands of metabolites with varying chemical properties in a biological sample presents unique challenges, or pitfalls. In this review, we address the various obstacles and corrective measures available in four major aspects associated with an untargeted metabolomics experiment: (1) experimental design, (2) pre-analytical (sample collection and preparation), (3) analytical (chromatography and detection), and (4) post-analytical (data processing).

Transplantation of wild-type mouse hematopoietic stem and progenitor cells ameliorates deficits in a mouse model of Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an incurable autosomal recessive neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin due to an intronic GAA-repeat expansion in the FXN gene. We report the therapeutic efficacy of transplanting wild-type mouse hematopoietic stem and progenitor cells (HSPCs) into the YG8R mouse model of FRDA. In the HSPC-transplanted YG8R mice, development of muscle weakness and locomotor deficits was abrogated as was degeneration of large sensory neurons in the dorsal root ganglia (DRGs) and mitochondrial capacity was improved in brain, skeletal muscle, and heart. Transplanted HSPCs engrafted and then differentiated into microglia in the brain and spinal cord and into macrophages in the DRGs, heart, and muscle of YG8R FRDA mice. We observed the transfer of wild-type frataxin and Cox8 mitochondrial proteins from HSPC-derived microglia/macrophages to FRDA mouse neurons and muscle myocytes in vivo. Our results show the HSPC-mediated phenotypic rescue of FRDA in YG8R mice and suggest that this approach should be investigated further as a strategy for treating FRDA.

Maternal obesity reduces oxidative capacity in fetal skeletal muscle of Japanese macaques.

Maternal obesity is proposed to alter the programming of metabolic systems in the offspring, increasing the risk for developing metabolic diseases; however, the cellular mechanisms remain poorly understood. Here, we used a nonhuman primate model to examine the impact of a maternal Western-style diet (WSD) alone, or in combination with obesity (Ob/WSD), on fetal skeletal muscle metabolism studied in the early third trimester. We find that fetal muscle responds to Ob/WSD by upregulating fatty acid metabolism, mitochondrial complex activity, and metabolic switches (CPT-1, PDK4) that promote lipid utilization over glucose oxidation. Ob/WSD fetuses also had reduced mitochondrial content, diminished oxidative capacity, and lower mitochondrial efficiency in muscle. The decrease in oxidative capacity and glucose metabolism was persistent in primary myotubes from Ob/WSD fetuses despite no additional lipid-induced stress. Switching obese mothers to a healthy diet prior to pregnancy did not improve fetal muscle mitochondrial function. Lastly, while maternal WSD alone led only to intermediary changes in fetal muscle metabolism, it was sufficient to increase oxidative damage and cellular stress. Our findings suggest that maternal obesity or WSD, alone or in combination, leads to programmed decreases in oxidative metabolism in offspring muscle. These alterations may have important implications for future health.

Diagnosis and Monitoring of Cystinosis Using Immunomagnetically Purified Granulocytes.

BACKGROUND: Cystine determination is a critical biochemical test for the diagnosis and therapeutic monitoring of the lysosomal storage disease cystinosis. The classical mixed-leukocyte cystine assay requires prompt specialized recovery/isolation following blood drawing, providing cystine concentrations normalized to total protein from assorted types of white blood cells, each with varying cystine content. METHODS: We present a new workflow for cystine determination using immunomagnetic granulocyte purification, and new reference ranges established from 47 patient and 27 obligate heterozygote samples assayed. Samples were collected in acid-citrate dextrose tubes and their stability was proven to allow for overnight shipping before analysis. Cystine was quantified by LC-MS/MS. RESULTS: The new method was reproducible (<15% root mean square error) and specific, assaying purified granulocytes from blood samples that no longer required immediate preparation and therefore allowing for up to 30 h before processing. There was a nearly a 2-fold increase in the therapeutic target (1.9 nmol half-cystine/mg protein) range, established using distributions of patient, obligate heterozygote, and control samples. The 2.5-97.5 percentile ranges (-2 SD to +2 SD around mean) for these cohorts were 0.67-6.05 nmol/mg protein for patients, 0.33-1.35 nmol/mg protein for obligate heterozygotes, and 0.09-0.35 nmol/mg protein for controls. CONCLUSIONS: The intracellular cystine determination method using immunopurified granulocytes followed by LC-MS/MS analysis improves the inherent variability of mixed leukocyte analysis and eliminates the need for immediate sample preparation following blood draw.

p300 is not required for metabolic adaptation to endurance exercise training.

The acetyltransferase, E1a-binding protein (p300), is proposed to regulate various aspects of skeletal muscle development, metabolism, and mitochondrial function,viaits interaction with numerous transcriptional regulators and other proteins. Remarkably, however, the contribution of p300 to skeletal muscle function and metabolism,in vivo, is poorly understood. To address this, we used Cre-LoxP methodology to generate mice with skeletal muscle-specific knockout of E1a-binding protein (mKO). mKO mice were indistinguishable from their wild-type/floxed littermates, with no differences in lean mass, skeletal muscle structure, fiber type, respirometry flux, or metabolites of fatty acid and amino acid metabolism.Ex vivomuscle function in extensor digitorum longus and soleus muscles, including peak stress and time to fatigue, as well asin vivorunning capacity were also comparable. Moreover, expected adaptations to a 20 d voluntary wheel running regime were not compromised in mKO mice. Taken together, these findings demonstrate that p300 is not required for the normal development or functioning of adult skeletal muscle, nor is it required for endurance exercise-mediated mitochondrial adaptations.-LaBarge, S. A., Migdal, C. W., Buckner, E. H., Okuno, H., Gertsman, I., Stocks, B., Barshop, B. A., Nalbandian, S. R., Philp, A., McCurdy, C. E., Schenk, S. p300 is not required for metabolic adaptation to endurance exercise training.

Perturbations of tyrosine metabolism promote the indolepyruvate pathway via tryptophan in host and microbiome.

The drug nitisinone (NTBC) is used to treat tyrosinemia type I, and more recently has been also used for the treatment of another disorder of tyrosine metabolism, alkaptonuria. While studying the dose effects of NTBC treatment on alkaptonuria, untargeted metabolomics revealed perturbations in a completely separate pathway, that of tryptophan metabolism. Significant elevations in several indolic compounds associated with the indolepyruvate pathway of tryptophan metabolism were present in NTBC-treated patient sera and correlated with elevations of an intermediate of tyrosine metabolism. Indolic compounds of this pathway have long been associated with commensal bacterial and plant metabolism. These exogenous sources of indoles have been more recently implicated in affecting mammalian cell function and disease. We studied the correlation of these indolic compounds in other disorders of tyrosine metabolism including tyrosinemia types I and II as well as transient tyrosinemia, and demonstrated that 4-hydroxyphenylpyruvate (4-HPP) was directly responsible for the promotion of this pathway. We then investigated the regulation of the indolepyruvate pathway and the role of 4-HPP further in both mammalian cells and intestinal microbial cultures. We demonstrated that several of the indolic products, including indolepyruvate and indolelactate, were in fact generated by human cell metabolism, while the downstream indole metabolite, indolecarboxaldehyde, was produced exclusively by microbial cultures of human gut flora. This study describes a symbiotic perturbation in host and microbiome tryptophan metabolism in response to elevations related to defects of tyrosine metabolism and concomitant drug treatment.

Metabolic Effects of Increasing Doses of Nitisinone in the Treatment of Alkaptonuria.

Alkaptonuria is an autosomal recessive disease involving a deficiency of the enzyme homogentisate dioxygenase, which is involved in the tyrosine degradation pathway. The enzymatic deficiency results in high concentrations of homogentisic acid (HGA), which results in orthopedic and cardiac complications, among other symptoms. Nitisinone (NTBC) has been shown to effectively treat alkaptonuria by blocking the conversion of 4-hydroxyphenylpyruvate to HGA, but there have been concerns that using doses higher than about 2 mg/day could cause excessively high levels of tyrosine, resulting in crystal deposition and corneal pathology. We have enrolled seven patients in a study to determine whether higher doses of NTBC were effective at further reducing HGA levels while maintaining tyrosine at acceptable levels. Patients were given varying doses of NTBC (ranging from 2 to 8 mg/day) over the course of between 0.5 and 3.5 years. Urine HGA, plasma tyrosine levels, and plasma NTBC were then measured longitudinally at various doses. We found that tyrosine concentrations plateaued and did not reach significantly higher levels as NTBC doses were increased above 2 mg/day, while a significant drop in HGA continued from 2 to 4 mg/day, with no significant changes at higher doses. We also demonstrated using untargeted metabolomics that elevations in tyrosine from treatment resulted in proportional elevations in alternative tyrosine metabolic products, that of N-acetyltyrosine and γ-glutamyltyrosine.

Validation of a dual LC-HRMS platform for clinical metabolic diagnosis in serum, bridging quantitative analysis and untargeted metabolomics.

Mass spectrometry-based metabolomics is a rapidly growing field in both research and diagnosis. Generally, the methodologies and types of instruments used for clinical and other absolute quantification experiments are different from those used for biomarkers discovery and untargeted analysis, as the former requires optimal sensitivity and dynamic range, while the latter requires high resolution and high mass accuracy. We used a Q-TOF mass spectrometer with two different types of pentafluorophenyl (PFP) stationary phases, employing both positive and negative ionization, to develop and validate a hybrid quantification and discovery platform using LC-HRMS. This dual-PFP LC-MS platform quantifies over 50 clinically relevant metabolites in serum (using both MS and MS/MS acquisitions) while simultaneously collecting high resolution and high mass accuracy full scans to monitor all other co-eluting non-targeted analytes. We demonstrate that the linearity, accuracy, and precision results for the quantification of a number of metabolites, including amino acids, organic acids, acylcarnitines and purines/pyrimidines, meets or exceeds normal bioanalytical standards over their respective physiological ranges. The chromatography resolved highly polar as well as hydrophobic analytes under reverse-phase conditions, enabling analysis of a wide range of chemicals, necessary for untargeted metabolomics experiments. Though previous LC-HRMS methods have demonstrated quantification capabilities for various drug and small molecule compounds, the present study provides an HRMS quant/qual platform tailored to metabolic disease; and covers a multitude of different metabolites including compounds normally quantified by a combination of separate instrumentation.

Mechanics of bacteriophage maturation.

Capsid maturation with large-scale subunit reorganization occurs in virtually all viruses that use a motor to package nucleic acid into preformed particles. A variety of ensemble studies indicate that the particles gain greater stability during this process, however, it is unknown which material properties of the fragile procapsids change. Using Atomic Force Microscopy-based nano-indentation, we study the development of the mechanical properties during maturation of bacteriophage HK97, a λ-like phage of which the maturation-induced morphological changes are well described. We show that mechanical stabilization and strengthening occurs in three independent ways: (i) an increase of the Young's modulus, (ii) a strong rise of the capsid's ultimate strength, and (iii) a growth of the resistance against material fatigue. The Young's modulus of immature and mature capsids, as determined from thin shell theory, fit with the values calculated using a new multiscale simulation approach. This multiscale calculation shows that the increase in Young's modulus isn't dependent on the crosslinking between capsomers. In contrast, the ultimate strength of the capsids does increase even when a limited number of cross-links are formed while full crosslinking appears to protect the shell against material fatigue. Compared to phage λ, the covalent crosslinking at the icosahedral and quasi threefold axes of HK97 yields a mechanically more robust particle than the addition of the gpD protein during maturation of phage λ. These results corroborate the expected increase in capsid stability and strength during maturation, however in an unexpected intricate way, underlining the complex structure of these self-assembling nanocontainers.

The Prohead-I structure of bacteriophage HK97: implications for scaffold-mediated control of particle assembly and maturation.

Virus capsid assembly requires recruiting and organizing multiple copies of protein subunits to form a closed shell for genome packaging that leads to infectivity. Many viruses encode scaffolding proteins to shift the equilibrium toward particle formation by promoting intersubunit interactions and stabilizing assembly intermediates. Bacteriophage HK97 lacks an explicit scaffolding protein, but the capsid protein (gp5) contains a scaffold-like N-terminal segment termed the delta domain. When gp5 is expressed in Escherichia coli, the delta domain guides 420 copies of the subunit into a procapsid with T=7 laevo icosahedral symmetry named Prohead-I. Prohead-I can be disassembled and reassembled under mild conditions and it cannot mature further. When the virally encoded protease (gp4) is coexpressed with gp5, it is incorporated into the capsid and digests the delta domain followed by autoproteolysis to produce the metastable Prohead-II. Prohead-I(+P) was isolated by coexpressing gp5 and an inactive mutant of gp4. Prohead-I and Prohead-I(+P) were compared by biochemical methods, revealing that the inactive protease stabilized the capsid against disassembly by chemical or physical stress. The crystal structure of Prohead-I(+P) was determined at 5.2 Å resolution, and distortions were observed in the subunit tertiary structures similar to those observed previously in Prohead-II. Prohead-I(+P) differed from Prohead-II due to the presence of the delta domain and the resulting repositioning of the N-arms, explaining why Prohead-I can be reversibly dissociated and cannot mature. Low-resolution X-ray data enhanced the density of the relatively dynamic delta domains, revealing their quaternary arrangement and suggesting how they drive proper assembly.

Critical salt bridges guide capsid assembly, stability, and maturation behavior in bacteriophage HK97.

HK97 is a double-stranded DNA bacteriophage that undergoes dramatic conformational changes during viral capsid maturation and for which x-ray structures, at near atomic resolution, of multiple intermediate and mature capsid states are available. Both amide H/(2)H exchange and crystallographic comparisons between the pre-expanded Prohead II particles and the expanded Head II of bacteriophage HK97 revealed quaternary interactions that remain fixed throughout maturation and appear to maintain intercapsomer integrity at all quasi- and icosahedral 3-fold axes. These 3-fold staples are formed from Arg and Glu residues and a metal binding site. Mutations of either Arg-347 or Arg-194 or a double mutation of E344Q and E363A resulted in purification of the phage in capsomer form (hexamers and pentamers). Mutants that did assemble had both decreased thermal stability and decreased in vitro expansion rates. Amide H/(2)H exchange mass spectrometry showed that in the wild type capsid some subunits had a bent "spine" helix (highly exchanging), whereas others were straight (less exchanging). Similar analysis of the never assembled mutant capsomers showed uniform amide exchange in all of these that was higher than that of the straight spine helices (characterized in more mature intermediates), suggesting that the spine helix is somewhat bent prior to capsid assembly. The result further supports a previously proposed mechanism for capsid expansion in which the delta domains of each subunit induce a high energy intermediate conformation, which now appears to include a bent helix during capsomer assembly.

HK97 maturation studied by crystallography and H/2H exchange reveals the structural basis for exothermic particle transitions.

HK97 is an exceptionally amenable system for characterizing major conformational changes associated with capsid maturation in double-stranded DNA bacteriophage. HK97 undergoes a capsid expansion of approximately 20%, accompanied by major subunit rearrangements during genome packaging. A previous 3.44-A-resolution crystal structure of the mature capsid Head II and cryo-electron microscopy studies of other intermediate expansion forms of HK97 suggested that, primarily, rigid-body movements facilitated the maturation process. We recently reported a 3.65-A-resolution structure of the preexpanded particle form Prohead II (P-II) and found that the capsid subunits undergo significant refolding and twisting of the tertiary structure to accommodate expansion. The P-II study focused on major twisting motions in the P-domain and on refolding of the spine helix during the transition. Here we extend the crystallographic comparison between P-II and Head II, characterizing the refolding events occurring in each of the four major domains of the capsid subunit and their effect on quaternary structure stabilization. In addition, hydrogen/deuterium exchange, coupled to mass spectrometry, was used to characterize the structural dynamics of three distinct capsid intermediates: P-II, Expansion Intermediate, and the nearly mature Head I. Differences in the solvent accessibilities of the seven quasi-equivalent capsid subunits, attributed to differences in secondary and quaternary structures, were observed in P-II. Nearly all differences in solvent accessibility among subunits disappear after the first transition to Expansion Intermediate. We show that most of the refolding is coupled to this transformation, an event associated with the transition from asymmetric to symmetric hexamers.

An unexpected twist in viral capsid maturation.

Lambda-like double-stranded (ds) DNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm of pressure during genome packaging. The extensive integration between subunits in capsids requires the formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Although various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage. Here we present a procapsid X-ray structure at 3.65 A resolution, termed prohead II, of the lambda-like bacteriophage HK97, the mature capsid structure of which was previously solved to 3.44 A (ref. 2). A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and hydrogen/deuterium exchange data presented here demonstrate that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and beta-sheet regions. We also identified subunit interactions at each three-fold axis of the capsid that are maintained throughout maturation. The interactions sustain capsid integrity during subunit refolding and provide a fixed hinge from which subunits undergo rotational and translational motions during maturation. Previously published calorimetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90 kJ mol(-1) of energy. We propose that the major tertiary changes presented in this study reveal a structural basis for an exothermic maturation process probably present in many dsDNA bacteriophage and possibly viruses such as herpesvirus, which share the HK97 subunit fold.