Active Human Cytomegalovirus Protease Is a Dimer

Darke, Paul L.; Cole, James L.; Waxman, Lloyd; Hall, Dawn L.; Sardana, Mohinder K.; Kuo, Lawrence C.

doi:10.1074/jbc.271.13.7445

Cited by 97 publications

(170 citation statements)

References 13 publications

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“…The dilution method has been used to study the dimer-monomer equilibrium of several herpes viral proteases with K d values in the nanomolar range (33)(34)(35). If the monomeric DPP-IV has a very low activity as observed for monomeric H750A and H750E, dilution of the protease to a concentration near or below the K d value might result in the formation of low activity monomeric DPP-IV.…”

Section: Fig 5 Sedimentation Velocity Analysis Of Dpp-iv Proteinsmentioning

confidence: 99%

One Site Mutation Disrupts Dimer Formation in Human DPP-IV Proteins

Chien

Huang

Chou

et al. 2004

Journal of Biological Chemistry

103

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DPP-IV is a prolyl dipeptidase, cleaving the peptide bond after the penultimate proline residue. It is an important drug target for the treatment of type II diabetes. DPP-IV is active as a dimer, and monomeric DPP-IV has been speculated to be inactive. In this study, we have identified the C-terminal loop of DPP-IV, highly conserved among prolyl dipeptidases, as essential for dimer formation and optimal catalysis. The conserved residue His 750 on the loop contributes significantly for dimer stability. We have determined the quaternary structures of the wild type, H750A, and H750E mutant enzymes by several independent methods including chemical crosslinking, gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Wild-type DPP-IV exists as dimers both in the intact cell and in vitro after purification from human semen or insect cells. The H750A mutation results in a mixture of DPP-IV dimer and monomer. H750A dimer has the same kinetic constants as those of the wild type, whereas the H750A monomer has a 60-fold decrease in k cat . Replacement of His 750 with a negatively charged Glu (H750E) results in nearly exclusive monomers with a 300-fold decrease in catalytic activity. Interestingly, there is no dynamic equilibrium between the dimer and the monomer for all forms of DPP-IVs studied here. This is the first study of the function of the C-terminal loop as well as monomeric mutant DPP-IVs with respect to their enzymatic activities. The study has important implications for the discovery of drugs targeted to the dimer interface.

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Section: Fig 5 Sedimentation Velocity Analysis Of Dpp-iv Proteinsmentioning

confidence: 99%

One Site Mutation Disrupts Dimer Formation in Human DPP-IV Proteins

Chien

Huang

Chou

et al. 2004

Journal of Biological Chemistry

103

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“…A central part of the regulation of assemblin activity is a monomer-dimer equilibrium [37]. The dimer is weakly associated with dissociation constants (K D ) in the micromolar range [79].…”

Section: Regulation Of Activitymentioning

confidence: 99%

“…It is the active species, while the monomer is almost inactive. The equilibrium is shifted towards the dimer by higher concentrations of assemblin, salts and co-solvents like glycerol [37,75,79]. Dimerization induces refolding of a loop (residues 162 to 171) near the active site.…”

Section: Regulation Of Activitymentioning

confidence: 99%

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Assemblins as maturational proteases in herpesviruses

Zühlsdorf

Hinrichs

2017

Journal of General Virology

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During assembly of herpesvirus capsids, a protein scaffold self-assembles to ring-like structures forming the scaffold of the spherical procapsids. Proteolytic activity of the herpesvirus maturational protease causes structural changes that result in angularization of the capsids. In those mature icosahedral capsids, the packaging of viral DNA into the capsids can take place. The strictly regulated protease is called assemblin. It is inactive in its monomeric state and activated by dimerization. The structures of the dimeric forms of several assemblins from all herpesvirus subfamilies have been elucidated in the last two decades. They revealed a unique serine-protease fold with a catalytic triad consisting of a serine and two histidines. Inhibitors that disturb dimerization by binding to the dimerization area were found recently. Additionally, the structure of the monomeric form of assemblin from pseudorabies virus and some monomer-like structures of Kaposi's sarcoma-associated herpesvirus assemblin were solved. These findings are the proof-of-principle for the development of new anti-herpesvirus drugs. Therefore, the most important information on this fascinating and unique class of proteases is summarized here.

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“…Lightscattering studies showed that the protease was monodisperse in solution and existed as dimers. This observation was made before the appearances of published reports showing the presence of the dimer and suggesting that the dimer is the active form of the enzyme (Darke et al, 1996;Margosiak, Vanderpool, Sisson, Pinko & Kan, 1996). Initial crystallization conditions were found with the sparse-matrix sampling technique (Jancarik & Kim, 1991), using a commercial kit (Hampton Research).…”

Section: Treatment Of Crystalsmentioning

confidence: 99%

Experiences from the Structure Determination of Human Cytomegalovirus Protease

Tong

Qian²,

Davidson³

et al. 1997

Acta Cryst D

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Several obstacles were encountered and overcome during the structure determination of human cytomegalovirus protease. Dehydration of crystals, by exposing them to higher concentrations of the precipitant, reduced the mosaicity of the crystals and may have also resolved their microscopic twinning. The initial phase information was obtained with the selenomethionyl multiple-wavelength anomalous diffraction technique. However, site-specific mutagenesis was required to introduce extra Met residues into the protease. The phase information had to be improved by non-crystallographic symmetry averaging, initially among three 'crystal forms'. A change in the composition of the artificial mother liquor led to a significant improvement, from 3.0 and 2.0A resolution, in the diffraction quality of the crystals. The experiences reported here may prove useful to structure determination of other proteins.

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Active Human Cytomegalovirus Protease Is a Dimer

Cited by 97 publications

References 13 publications

One Site Mutation Disrupts Dimer Formation in Human DPP-IV Proteins

One Site Mutation Disrupts Dimer Formation in Human DPP-IV Proteins

Assemblins as maturational proteases in herpesviruses

Experiences from the Structure Determination of Human Cytomegalovirus Protease

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