Previously, a charge balance hypothesis was proposed to explain hepatitis B virus (HBV) capsid stability, assembly, RNA encapsidation, and DNA replication. This hypothesis emphasized the importance of a balanced electrostatic interaction between the positive charge from the arginine-rich domain (ARD) of the core protein (HBc) and the negative charge from the encapsidated nucleic acid. It remains unclear if any of the negative charge involved in this electrostatic interaction could come from the HBc protein per se, in addition to the encapsidated nucleic acid. HBc ARD IV mutant 173GG and ARD II mutant 173RR/R157A/R158A are arginine deficient and replication defective. Not surprisingly, the replication defect of ARD IV mutant 173GG can be rescued by restoring positively charged amino acids at the adjacent positions 174 and 175. However, most interestingly, it can be at least partially rescued by reducing negatively charged residues in the assembly domain, such as by glutamic acid-to-alanine (E-to-A) substitutions at position 46 or 117 and to a much lesser extent at position 113. Similar results were obtained for ARD II mutant 173RR/R157A/R158A. These amino acids are located on the inner surfaces of HBc icosahedral particles, and their acidic side chains point toward the capsid interior. For HBV DNA synthesis, the relative amount of positive versus negative charge in the electrostatic interactions is more important than the absolute amount of positive or negative charge. These results support the concept that balanced electrostatic interaction is important during the viral life cycle. Human hepatitis B virus (HBV)is an important human pathogen (5, 27, 34) that can replicate via an RNA intermediate (31, 33). Wild-type (WT) HBV core protein (HBc) is 183 amino acids long (adr and ayw subtypes) and consists of two distinct domains connected by a hinge region. The assembly domain spans amino acids 1 to 140, and the arginine-rich domain (ARD) spans amino acids 150 to 183. The ARD of HBc 150-183 is not required for capsid assembly in Escherichia coli (4,8,10,21,35). During nucleocapsid (capsid) formation, the HBc protein assembles into an icosahedral particle via a dimer intermediate (32). The ARD of HBc is known to be capable of binding to nucleic acids (12,25). Serine phosphorylation at the C terminus of HBc is known to be important for RNA encapsidation, DNA synthesis, and virion secretion (2,11,14,15,17,19,24,26,39,40). To date, there is no structural information available for the C terminus of HBc in capsids (32). The 4-helix bundle structure of HBc capsids is based on a C-terminally truncated capsid protein, HBc149 (6, 7, 36). Our research progress in the study of HBV biology has been hampered due to the lack of structural information about the HBc C-terminal tail, which plays an important regulatory role throughout the life cycle of HBV.Recently, we proposed a hypothesis that "charge balance" could be important for HBV capsid stability, assembly, RNA encapsidation, and DNA replication (17). This hypothesis postulat...

Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and ␣-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and ␣-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric ␣-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of ␣-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.

Heme oxygenase-1 (HO-1), a stress-inducible enzyme anchored in the endoplasmic reticulum (ER) by a single transmembrane segment (TMS) located at the C terminus, interacts with NADPH cytochrome P450 reductase and biliverdin reductase to catalyze heme degradation to biliverdin and its metabolite, bilirubin. Previous studies suggested that HO-1 functions as a monomer. Using chemical cross-linking, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments, here we showed that HO-1 forms dimers/ oligomers in the ER. However, oligomerization was not observed with a truncated HO-1 lacking the C-terminal TMS (amino acids 266 -285), which exhibited cytosolic and nuclear localization, indicating that the TMS is essential for the selfassembly of HO-1 in the ER. To identify the interface involved in the TMS-TMS interaction, residue Trp-270, predicted by molecular modeling as a potential interfacial residue of TMS ␣-helices, was mutated, and the effects on protein subcellular localization and activity assessed. The results showed that the W270A mutant was present exclusively in the ER and formed oligomers with similar activity to those of the wild type HO-1. Interestingly, the W270N mutant was localized not only in the ER, but also in the cytosol and nucleus, suggesting it is susceptible to proteolytic cleavage. Moreover, the microsomal HO activity of the W270N mutant was significantly lower than that of the wild type. The W270N mutation appears to interfere with the oligomeric state, as revealed by a lower FRET efficiency. Collectively, these data suggest that oligomerization, driven by TMS-TMS interactions, is crucial for the stabilization and function of HO-1 in the ER.Heme oxygenase (HO) 3 catalyzes the NADPH cytochrome P450 reductase-dependent oxidative degradation of cellular heme to biliverdin, carbon monoxide (CO), and free iron (1, 2). Biliverdin is subsequently converted to bilirubin by biliverdin reductase in the cytosol. Two HO isoforms have been identified in mammalian systems. HO-1 is a 288 amino acid protein and is expressed at high amounts in a variety of pathological conditions associated with cellular stress. There is compelling evidence that HO-1 induction represents an important cytoprotective defense mechanism against oxidative insults by virtue of the anti-oxidant properties of the bilirubin and the anti-inflammatory effect of the CO produced (2). HO-1 is anchored in the endoplasmic reticulum (ER) through a single transmembrane segment (TMS) located at the C terminus, while the rest of the molecule is cytoplasmic (3). HO-1 is sensitive to proteolytic cleavage (4), and it was recently shown that HO-1 can be proteolytically cleaved from the ER and translocated to the nucleus under certain stress conditions (5). Although the catalytic site in the cytoplasmic domain remains intact, the activity of soluble HO-1 is drastically reduced (5), indicating that ER localization is important for its full enzymatic function.Self-assembly to form dimers and higher oligomers is a common pheno...

Dipeptidyl peptidase IV (DPP-IV) is a drug target in the treatment of human type II diabetes. It is a type II membrane protein with a single transmembrane domain (TMD) anchoring the extracellular catalytic domain to the membrane. DPP-IV is active as a dimer, with two dimer interacting surfaces located extracellularly. In this study, we demonstrate that the TM of DPP-IV promotes DPP-IV dimerization and rescues monomeric DPP-IV mutants into partial dimers, which is specific and irreplaceable by TMs of other type II membrane proteins. By bioluminescence resonance energy transfer (BRET) and peptide electrophoresis, we found that the TM domain of DPP-IV is dimerized in mammalian cells and in vitro. The TM dimer interaction is very stable, based on our results with TM site-directed mutagenesis. None of the mutations, including the introduction of two prolines, resulted in their complete disruption to monomers. However, these TM proline mutations result in a significant reduction of DPP-IV enzymatic activity, comparable to what is found with mutations near the active site. A systematic analysis of TM structures deposited in the Protein Data Bank showed that prolines in the TM generally produce much bigger kinking angles than occur in nonproline-containing TMs. Thus, the proline-dependent reduction in enzyme activity may result from propagated conformational changes from the TM to the Abbreviations: Ala-Pro-AFC, H-Ala-Pro-7-amino-4-trifluoromethylcoumarin; BRET, bioluminescence resonance energy transfer; DPP, dipeptidyl peptidase; DPP-IV, dipeptidyl peptidase IV; EOM, enzyme overlay membrane; FBS, fetal bovine serum; GFP, green fluorescent protein; Gly-Pro-AMC, H-Gly-Pro-7-amino-4-methylcoumarin; GPCR, G-Protein coupled receptor; PBS, phosphate buffered saline; PDB, protein data bank; PDBTM, protein data bank of transmembrane proteins; PFO, perfluorooctanoate; Rluc, renilla luciferase; SI, sucrase isomaltase; TM, transmembrane; TMD, transmembrane domain; WT, wild-type.Additional Supporting Information may be found in the online version of this article.Kuei-Min Chung and Jai-Hong Cheng contributed equally to this article. Our results demonstrate that TM dimerization and conformation contribute significantly to the structure and activity of DPP-IV. Optimal enzymatic activity of DPP-IV requires an optimal interaction of all three dimer interfaces, including its TM.

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