RT Loop Flexibility Enhances the Specificity of Src Family SH3 Domains for HIV-1 Nef<sup>,</sup>

Arold, Stefan T.; O’Brien, Rita Cruise; Franken, P.; Strub, Marie‐Paule; Hoh, François; Dumas, Christian; Ladbury, John E.

doi:10.1021/bi980989q

Cited by 117 publications

(134 citation statements)

References 47 publications

Supporting

Mentioning

116

Contrasting

Unclassified

Order By: Relevance

“…The significant difference in the conformation of the RT-loop may be the result of a rearrangement occurring upon peptide binding, because this region is directly involved in the binding reaction. This is consistent with several other studies that have shown a role for RT-loop flexibility in binding (34,35). The ArkA peptide, which binds in a class II orientation (2, 3), contains an N-terminal left-handed polyproline type II (PPII) helix and a C-terminal 3 10 helix (Fig.…”

Section: Structural Studies Of Abpsh3⅐peptidesupporting

confidence: 91%

Structural, Functional, and Bioinformatic Studies Demonstrate the Crucial Role of an Extended Peptide Binding Site for the SH3 Domain of Yeast Abp1p

Stollar

Garcı́a

Chong

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

SH3 domains, which are among the most frequently occurring protein interaction modules in nature, bind to peptide targets ranging in length from 7 to more than 25 residues. Although the bulk of studies on the peptide binding properties of SH3 domains have focused on interactions with relatively short peptides (less than 10 residues), a number of domains have been recently shown to require much longer sequences for optimal binding affinity. To gain greater insight into the binding mechanism and biological importance of interactions between an SH3 domain and extended peptide sequences, we have investigated interactions of the yeast Abp1p SH3 domain (AbpSH3) with several physiologically relevant 17-residue target peptide sequences. To obtain a molecular model for AbpSH3 interactions, we solved the structure of the AbpSH3 bound to a target peptide from the yeast actin patch kinase, Ark1p. Peptide target complexes from binding partners Scp1p and Sjl2p were also characterized, revealing that the AbpSH3 uses a common extended interface for interaction with these peptides, despite K d values for these peptides ranging from 0.3 to 6 M. Mutagenesis studies demonstrated that residues across the whole 17-residue binding site are important both for maximal in vitro binding affinity and for in vivo function. Sequence conservation analysis revealed that both the AbpSH3 and its extended target sequences are highly conserved across diverse fungal species as well as higher eukaryotes. Our data imply that the AbpSH3 must bind extended target sites to function efficiently inside the cell.Many protein interactions within signaling pathways are mediated by small modular domains, which are found within larger proteins (1). SH3 domains are one of the most frequently occurring of these protein-protein interaction modules in eukaryotic cells. These domains are ϳ60-residue ␤-sheet proteins that have been generally observed to bind to short proline-rich peptides containing the core consensus sequences ϩXXPXXP (class I) or PXXPXϩ (class II), where X can be a variety of residues, and ϩ is a Lys or Arg residue (2-4). SH3 domains often bind peptides with modest affinities (5-100 M (5)), and many SH3 domains appear to possess low specificity, binding to various PXXP-containing peptides with similar affinities (6 -10). These observations have led to the establishment of a "promiscuous model," which postulates that the signaling specificity of pathways depends primarily on factors other than the intrinsic binding properties of isolated SH3 domains (11), and that short peptide targets are likely sufficient for SH3 domain function. Arguing against this model, it has been shown that some SH3 domains require an extended target peptide (12-30 residues) to achieve maximal binding affinity (12-15). These results imply that the intrinsic specificity of SH3 domains may indeed play a significant role. The growing realization of the importance of interactions between SH3 domains and extended peptides provides the motivation for further studies to investigat...

show abstract

Section: Structural Studies Of Abpsh3⅐peptidesupporting

confidence: 91%

Structural, Functional, and Bioinformatic Studies Demonstrate the Crucial Role of an Extended Peptide Binding Site for the SH3 Domain of Yeast Abp1p

Stollar

Garcı́a

Chong

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…3). As the hydrophobic pocket of HIV-1 Nef is determining its high affinity for Hck SH3 (K d ϭ 200 -600 nM) (7,8), the introduction of charged residues should considerably lower the affinity of SIV/HIV-2 Nef for this SH3 domain.…”

Section: Resultsmentioning

confidence: 99%

“…5, a and b). This may be explained by the difference in flexibility of the RT-loop of these SH3 domains in their free state (8). The binding to HIV-1 Nef requires breaking of fewer hydrogen bonds in the RT-loop of the Hck SH3 domain than in the RT-loop of Src or Fyn SH3 domains.…”

Section: Resultsmentioning

confidence: 99%

“…It has subsequently been shown that HIV-1 Nef binds with high affinity to the SH3 domains of the Src family kinases Hck and Lyn, whereas only with a modest affinity to SH3 domains of other members of the Src family (Lck, Fyn, and Src) (7,8). However, despite the PXXP motif being strictly conserved in all Nef proteins, an HIV-2 Nef allele has been reported to have lost the high affinity for Hck SH3 (9).…”

mentioning

confidence: 99%

See 1 more Smart Citation

HIV-2 and SIV Nef Proteins Target Different Src Family SH3 Domains than Does HIV-1 Nef because of a Triple Amino Acid Substitution

Collette¹,

Arold²,

Picard³

et al. 2000

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The nef gene is required for optimal viral spread of human and simian immunodeficiency viruses. However, the molecular mechanisms underlying the action of the Nef proteins may not be identical for all viral families. Here we investigate the interaction between the Nef protein of human and simian immunodeficiency viruses and SH3 domains from Src family kinases. Using the yeast two-hybrid system and immunoblotting we show that, in contrast to HIV-1 Nef, SIV and HIV-2 Nef poorly interact with Hck SH3 but bind to Src and Fyn SH3 domains. The molecular basis of these differences in SH3 targeting was revealed by sequence analysis and homology modeling of the putative SH3-Nef structures. Three amino acids (Trp-113, Thr-117, and Gln-118) that localize in a "hydrophobic pocket" implicated in SH3 binding of HIV-1 Nef, are systematically substituted in SIV/HIV-2 alleles (by Tyr, Glu, and Glu, respectively). We demonstrate that site-directed mutagenesis of these residues in SIV mac239 Nef suffices to restore Hck SH3 binding and co-immunoprecipitation with full-length Hck from transfected cells. Our findings identify fundamental mechanistic differences in targeting of Src family kinases by HIV and SIV Nef. The herein described mechanism of SH3 selection by Nef via a "pocket" proximal to the canonical proline-rich motif may be a common feature for SH3 recognition by their natural ligands.The nef gene is conserved among all primate lentiviruses and has been implicated in optimal viral replication and disease induction by HIV-1 1 and SIV (1, 2). Substantial sequence polymorphism has been detected among nef alleles from various cloned isolates of HIV and SIV, suggesting that Nef proteins may have subtype-specific functions or possess different strategies to perform the same task. However, four stretches of residues have been identified that were highly conserved among sequences derived from AIDS patients but also from among sequences of HIV-2 and SIV (3). One of these stretches includes a Pro-Xaa-Xaa-Pro motif, the canonical Src homology (SH) 3 domain binding consensus, indicating that SH3 binding is a common feature of all types of Nef alleles.An intact PXXP motif is required for the potential of HIV-1 Nef to enhance viral growth and infectivity in cell cultures (4 -6). It has subsequently been shown that HIV-1 Nef binds with high affinity to the SH3 domains of the Src family kinases Hck and Lyn, whereas only with a modest affinity to SH3 domains of other members of the Src family (Lck, Fyn, and Src) (7,8). However, despite the PXXP motif being strictly conserved in all Nef proteins, an HIV-2 Nef allele has been reported to have lost the high affinity for Hck SH3 (9). Controversial data has further been collected for the requirement of the proline-rich motif for AIDS disease progression in infected macaques (10, 11). Especially, it was reported that a PXXP to AXXA mutated SIV mac239 protein could still bind to Src kinase (10), suggesting that SIV might have different strategies of interacting with the Src family of protein tyro...

show abstract

“…Nef has been found to interact with several signaling molecules: with a serine kinase (17), with a distinct serine/threonine kinase, the Nef-associated kinase (NAK) identified as a member of the p21-activated kinase (PAK) family (18 -21), with members of the Src-family of tyrosine kinases, notably Lck (17,(22)(23)(24), Hck (25), Lyn, (26), Fyn (27), and with mitogen-activated protein kinase (MAPK) (23), c-Raf-1 (28), p53 (29), and protein kinase C (30).…”

mentioning

confidence: 99%

Exogenous Nef Protein Activates NF-κB, AP-1, and c-Jun N-Terminal Kinase and Stimulates HIV Transcription in Promonocytic Cells

Varin¹,

Manna²,

Quivy³

et al. 2003

Journal of Biological Chemistry

102

111

View full text Add to dashboard Cite

RT Loop Flexibility Enhances the Specificity of Src Family SH3 Domains for HIV-1 Nef^,

Cited by 117 publications

References 47 publications

Structural, Functional, and Bioinformatic Studies Demonstrate the Crucial Role of an Extended Peptide Binding Site for the SH3 Domain of Yeast Abp1p

Structural, Functional, and Bioinformatic Studies Demonstrate the Crucial Role of an Extended Peptide Binding Site for the SH3 Domain of Yeast Abp1p

HIV-2 and SIV Nef Proteins Target Different Src Family SH3 Domains than Does HIV-1 Nef because of a Triple Amino Acid Substitution

Exogenous Nef Protein Activates NF-κB, AP-1, and c-Jun N-Terminal Kinase and Stimulates HIV Transcription in Promonocytic Cells

Contact Info

Product

Resources

About

RT Loop Flexibility Enhances the Specificity of Src Family SH3 Domains for HIV-1 Nef,

Cited by 117 publications

References 47 publications

Structural, Functional, and Bioinformatic Studies Demonstrate the Crucial Role of an Extended Peptide Binding Site for the SH3 Domain of Yeast Abp1p

Structural, Functional, and Bioinformatic Studies Demonstrate the Crucial Role of an Extended Peptide Binding Site for the SH3 Domain of Yeast Abp1p

HIV-2 and SIV Nef Proteins Target Different Src Family SH3 Domains than Does HIV-1 Nef because of a Triple Amino Acid Substitution

Exogenous Nef Protein Activates NF-κB, AP-1, and c-Jun N-Terminal Kinase and Stimulates HIV Transcription in Promonocytic Cells

Contact Info

Product

Resources

About

RT Loop Flexibility Enhances the Specificity of Src Family SH3 Domains for HIV-1 Nef^,