The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding
<p>Abstract</p> <p>Background</p> <p>The ribonuclease H (RNase H) domains of retroviral reverse transcriptases play an essential role in the replication cycle of retroviruses. During reverse transcription of the viral genomic RNA, an RNA/DNA hybrid is created whose RNA...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2012-09-01
|
| Series: | Retrovirology |
| Subjects: | |
| Online Access: | http://www.retrovirology.com/content/9/1/73 |
| _version_ | 1818750177617379328 |
|---|---|
| author | Leo Berit Schweimer Kristian Rösch Paul Hartl Maximilian J Wöhrl Birgitta M |
| author_facet | Leo Berit Schweimer Kristian Rösch Paul Hartl Maximilian J Wöhrl Birgitta M |
| author_sort | Leo Berit |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>The ribonuclease H (RNase H) domains of retroviral reverse transcriptases play an essential role in the replication cycle of retroviruses. During reverse transcription of the viral genomic RNA, an RNA/DNA hybrid is created whose RNA strand needs to be hydrolyzed by the RNase H to enable synthesis of the second DNA strand by the DNA polymerase function of the reverse transcriptase. Here, we report the solution structure of the separately purified RNase H domain from prototype foamy virus (PFV) revealing the so-called C-helix and the adjacent basic loop, which both were suggested to be important in substrate binding and activity.</p> <p>Results</p> <p>The solution structure of PFV RNase H shows that it contains a mixed five-stranded β-sheet, which is sandwiched by four α-helices (A-D), including the C-helix, on one side and one α-helix (helix E) on the opposite side. NMR titration experiments demonstrate that upon substrate addition signal changes can be detected predominantly in the basic loop as well as in the C-helix. All these regions are oriented towards the bound substrate. In addition, signal intensities corresponding to residues in the B-helix and the active site decrease, while only minor or no changes of the overall structure of the RNase H are detectable upon substrate binding. Dynamic studies confirm the monomeric state of the RNase H domain. Structure comparisons with HIV-1 RNase H, which lacks the basic protrusion, indicate that the basic loop is relevant for substrate interaction, while the C-helix appears to fulfill mainly structural functions, i.e. positioning the basic loop in the correct orientation for substrate binding.</p> <p>Conclusions</p> <p>The structural data of PFV RNase H demonstrate the importance of the basic loop, which contains four positively charged lysines, in substrate binding and the function of the C-helix in positioning of the loop. In the dimeric full length HIV-1 RT, the function of the basic loop is carried out by a different loop, which also harbors basic residues, derived from the connection domain of the p66 subunit. Our results suggest that RNases H which are also active as separate domains might need a functional basic loop for proper substrate binding.</p> |
| first_indexed | 2024-12-18T04:15:32Z |
| format | Article |
| id | doaj.art-617d873805e040d3adf699af5ecb8e8e |
| institution | Directory Open Access Journal |
| issn | 1742-4690 |
| language | English |
| last_indexed | 2024-12-18T04:15:32Z |
| publishDate | 2012-09-01 |
| publisher | BMC |
| record_format | Article |
| series | Retrovirology |
| spelling | doaj.art-617d873805e040d3adf699af5ecb8e8e2022-12-21T21:21:21ZengBMCRetrovirology1742-46902012-09-01917310.1186/1742-4690-9-73The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate bindingLeo BeritSchweimer KristianRösch PaulHartl Maximilian JWöhrl Birgitta M<p>Abstract</p> <p>Background</p> <p>The ribonuclease H (RNase H) domains of retroviral reverse transcriptases play an essential role in the replication cycle of retroviruses. During reverse transcription of the viral genomic RNA, an RNA/DNA hybrid is created whose RNA strand needs to be hydrolyzed by the RNase H to enable synthesis of the second DNA strand by the DNA polymerase function of the reverse transcriptase. Here, we report the solution structure of the separately purified RNase H domain from prototype foamy virus (PFV) revealing the so-called C-helix and the adjacent basic loop, which both were suggested to be important in substrate binding and activity.</p> <p>Results</p> <p>The solution structure of PFV RNase H shows that it contains a mixed five-stranded β-sheet, which is sandwiched by four α-helices (A-D), including the C-helix, on one side and one α-helix (helix E) on the opposite side. NMR titration experiments demonstrate that upon substrate addition signal changes can be detected predominantly in the basic loop as well as in the C-helix. All these regions are oriented towards the bound substrate. In addition, signal intensities corresponding to residues in the B-helix and the active site decrease, while only minor or no changes of the overall structure of the RNase H are detectable upon substrate binding. Dynamic studies confirm the monomeric state of the RNase H domain. Structure comparisons with HIV-1 RNase H, which lacks the basic protrusion, indicate that the basic loop is relevant for substrate interaction, while the C-helix appears to fulfill mainly structural functions, i.e. positioning the basic loop in the correct orientation for substrate binding.</p> <p>Conclusions</p> <p>The structural data of PFV RNase H demonstrate the importance of the basic loop, which contains four positively charged lysines, in substrate binding and the function of the C-helix in positioning of the loop. In the dimeric full length HIV-1 RT, the function of the basic loop is carried out by a different loop, which also harbors basic residues, derived from the connection domain of the p66 subunit. Our results suggest that RNases H which are also active as separate domains might need a functional basic loop for proper substrate binding.</p>http://www.retrovirology.com/content/9/1/73Foamy virusRetroviral RNase HC-helixBasic loopNMRBasic protrusionSolution structureSubstrate binding |
| spellingShingle | Leo Berit Schweimer Kristian Rösch Paul Hartl Maximilian J Wöhrl Birgitta M The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding Retrovirology Foamy virus Retroviral RNase H C-helix Basic loop NMR Basic protrusion Solution structure Substrate binding |
| title | The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding |
| title_full | The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding |
| title_fullStr | The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding |
| title_full_unstemmed | The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding |
| title_short | The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding |
| title_sort | solution structure of the prototype foamy virus rnase h domain indicates an important role of the basic loop in substrate binding |
| topic | Foamy virus Retroviral RNase H C-helix Basic loop NMR Basic protrusion Solution structure Substrate binding |
| url | http://www.retrovirology.com/content/9/1/73 |
| work_keys_str_mv | AT leoberit thesolutionstructureoftheprototypefoamyvirusrnasehdomainindicatesanimportantroleofthebasicloopinsubstratebinding AT schweimerkristian thesolutionstructureoftheprototypefoamyvirusrnasehdomainindicatesanimportantroleofthebasicloopinsubstratebinding AT roschpaul thesolutionstructureoftheprototypefoamyvirusrnasehdomainindicatesanimportantroleofthebasicloopinsubstratebinding AT hartlmaximilianj thesolutionstructureoftheprototypefoamyvirusrnasehdomainindicatesanimportantroleofthebasicloopinsubstratebinding AT wohrlbirgittam thesolutionstructureoftheprototypefoamyvirusrnasehdomainindicatesanimportantroleofthebasicloopinsubstratebinding AT leoberit solutionstructureoftheprototypefoamyvirusrnasehdomainindicatesanimportantroleofthebasicloopinsubstratebinding AT schweimerkristian solutionstructureoftheprototypefoamyvirusrnasehdomainindicatesanimportantroleofthebasicloopinsubstratebinding AT roschpaul solutionstructureoftheprototypefoamyvirusrnasehdomainindicatesanimportantroleofthebasicloopinsubstratebinding AT hartlmaximilianj solutionstructureoftheprototypefoamyvirusrnasehdomainindicatesanimportantroleofthebasicloopinsubstratebinding AT wohrlbirgittam solutionstructureoftheprototypefoamyvirusrnasehdomainindicatesanimportantroleofthebasicloopinsubstratebinding |