Rac activation is regulated by the nucleotide guanosine 5'-triphosphate (GTP< >). Rac cycles between active GTP-bound and inactive GDP-bound confirmations. GTP-bound Rac is able to interact with downstream effector molecules, such as p67phox (Bishop et al, 2000). Guanosine nucelotide exchange factors (GEFs) regulate the transition from GDP-Rac to GTP-Rac. GTP-ase activating proteins (GAPs) facilitate GTP hydrolysis associated with the switch from GTP-Rac to GDP-Rac (Bishop et al, 2000). RhoGDI (gaunosine nucleotide dissociation factor) regulates Rac cycling between the GTP and GDP-bound states by sequestering GDP-Rac (Bishop et al, 2000).
The primary confirmational differences between GDP-bound and GTP-bound forms of Rac are localized to two surface loops, Switch I< > and Switch II< > (amino acids 26-45 and 59-74). Effector molecules must recognize structural discrepancies between these regions of GDP and GTP-bound Rac in order to bind Rac in the appropriate state (Bishop et al 2000). Rac interactions with p67phox induce confirmational changes in the effector protein which relay subsequent allosteric regulation of the NADPH oxidase complex (Bishop et al, 2000). Two Rac isoforms, Rac1 and Rac2, are expressed in neutrophils and macrophages. Both Rac1 and Rac2 are capable of binding p67phox and activating NADPH oxidase (Lapouge et al, 2000). Rac1 and Rac2 share 92% sequence homology; Switch I and Switch II are conserved between the two isoforms, with most sequence divergence occuring in the C-termini< > (Lapouge et al 2000). Rac1 crystallized with p67phox by Lapouge and colleagues (2000), Rac1-Q61L-GTP, was GTPase-deficient but contained an intact N-terminus< >, the region of the protein essential for interaction with p67phox. Removal of GTPase function reduced the flexibility of the Switch I region but did not affect affinity between p67phox and Rac1. III. p67phox p67phox is one of three cytosolic proteins activated in the assembly of NADPH oxidase complex. Upon activation by GTP-Rac1, p67phox translocates to the plasma membrane where it associates with other members of the NADPH oxidase complex. Binding affinity assays (Lapouge et al, 2000) established that the p67phox N-terminus contains all elements necessary for Rac binding. The p67phox fragment crystralized with Rac1 by Lapouge et al (2000) contains amino acids 1-203, the N-terminus of the protein. This fragment contains 9 alpha helices; eight of these helices were part of four TPR (tetrico peptide repeat) motifs. TPRs are degenerate 34 amino acid repeats that occur in tandem arrays and are involved in a variety of protein-protein interactions. TPR motifs are often found in scaffolding proteins in large complexes. The nine TPRs, each forming two antiparallel alpha helices, together create a nine alpha superhelical structure. This structure creates a groove in the TPR motifs that binds the C terminal of the third TPR helix (Lapouge et al, 2000). Please Reload Image. The p67phox TPR segment also contains a beta hairpin < >created by a 20 amino acid insert between the third and fourth TPR repeats. The beta hairpin is positioned by hydrogen bonds with His-69, Val-72, Glu-96, Leu-98, Ile-99, Glu-122, Val-123, and Leu-124. < > Positioning of the beta hairpin also relies on hydrogen bonds between Gly-113 and Arg-184 < > and between Glu-115andLys-181.< > IV. Rac1/p67phox Interface The p67phox regions necessary for Rac1 binding are contained within the N-terminus of p67phox (Lapouge et al). These binding interactions are localized to the p67phox TPR region beta hairpin structure and loops connecting TPR1 to TPR2, and TPR2 to TPR3. These contacts include both direct and water-mediated hydrogen bonds (lapouge et al, 2000). Please Reload Image. The beta hairpin insertion Asp-108p67contactsThr-25 of Rac1.< > Arg-102p67hydrogen bonds to Ala-159, Leu-160, Asn-26,and Ser-22 of Rac1. < > Asn-104p67hydrogen bonds to Asn-26Rac1andGln-162Rac1< >. The N terminus and C terminus of Rac1 are localized to the same area of the protein's 3D structure. Rac1 interactions with p67phox are localized to this region of the N and C termini, including Helix 1, the N terminal of Switch I, and the loop connecting beta chain 5 to alpha chain 5. Please Reload Image. These contacts include hydrogen bonds between Gly-30Rac1andAsp-67p67 < >and between Glu-31Rac1andSer-37p67< >. No contacts are made between p67phox and Rac Switch II or the insertion helix (Lapouge et al, 2000). V. Clinical Significance Mutations in any of the four NADPH oxidase subunits results in chronic granulomatous disease (CGD). CGD patients experience reoccuring infections as a result of phagocytic leukocytes unable to produce superoxide, a necessay component of defense against invading microorganisms (Leusen et al, 1996). The genetic basis of CGD can be classified into one of four genotypes. The majority of CGD pateints (60%) have X-linked mutations of the CYBB gene, which results in an abnormal B subunit in the cytochrome b558 component of NADPH oxidase complex (Leusen et al, 1996). Approximately 5% of CGD patients have an autosomal mutation of the CYBA gene, which encodes the alpha cytochrome subunit. Mutations of the NCFI gene encoding a 47kD component of the NADPH oxidase complex have been characterized in 30% of patients. Less than 5% of patients have CGD as a result of homozygous mutations in the NCF2 gene encoding for p67phox. These patients often express nonfunctional p67phox as a result of deletion of Lys-58. This deletion inhibits interactions between Rac1 and p67phox;consequently p67phox is unable to translocate to the plama membrane and subsequently unable to activate the NADPH oxidase complex. The patient characerized by Leusen and colleagues (1996) is heterozygous for two mutations, inlcuding a triplet deletion in the NCF2 sequence coding for p67phox. This mutation corresponds to Lys-58 deletion and a larger 11-13 kB deletion on the other p67phox allele. The patient's nonfunctional p67phox is unable to interact with Rac and therefore unable to translocate to the plasma membrane (Leusen et al, 1996). This patient's phenotype emphasizes the importance of Lys-58 in p67phox binding interactions with Rac and subsequent NADPH oxidase activation. VI. References Lapouge, K., S. Smith, P. Walker, S. Gamblin, S. Smerdon, K. Rittinger. Structure of the TPR domain of p67phox in complex with Rac-GTP. 2000. Molecular Cell, 6:899-907. Park, JB. Phagocytosis induces superoxide formation and apoptosis in macrophages. 2003. Experimental and Molecular Medicine, 35 (5): 325-335. Bishop, A., A. Hall. Rho GTPases and their effector proteins. 2000. Biochemical Journal, 348; 241-255. Diekmann, D., A. Abo, C. Johnston, A. Segal, A. Hall. Interactions of Rac with p67phox and regulation of phagocytic NADPH oxidase acitivity. 1994. Science, 265: 531-533. Abo, A., E. Pci, A. Hall, N. Totty, C. Teahan, A. Segal. Activation of the NADPH oxidase involved the small GTP-binding protein p21Rac1. 1991. Nature, 353: 668-670. Leusen J., A. de Klein, P. Hilarius, A. Ahlin, J. Palmblad, C. Smith, D. Diekmann, A. Hall, A. Vanhoeven, D. Roos. Disturbed interaction of p21-rac with mutated p67-phox causes chronic granulomatous disease. 1996. Journal of Experimental Medicine, 184: 1243-1249. *Special thanks to Gabe Schine for help with formating.*
Two Rac isoforms, Rac1 and Rac2, are expressed in neutrophils and macrophages. Both Rac1 and Rac2 are capable of binding p67phox and activating NADPH oxidase (Lapouge et al, 2000). Rac1 and Rac2 share 92% sequence homology; Switch I and Switch II are conserved between the two isoforms, with most sequence divergence occuring in the C-termini< > (Lapouge et al 2000). Rac1 crystallized with p67phox by Lapouge and colleagues (2000), Rac1-Q61L-GTP, was GTPase-deficient but contained an intact N-terminus< >, the region of the protein essential for interaction with p67phox. Removal of GTPase function reduced the flexibility of the Switch I region but did not affect affinity between p67phox and Rac1.
Binding affinity assays (Lapouge et al, 2000) established that the p67phox N-terminus contains all elements necessary for Rac binding. The p67phox fragment crystralized with Rac1 by Lapouge et al (2000) contains amino acids 1-203, the N-terminus of the protein. This fragment contains 9 alpha helices; eight of these helices were part of four TPR (tetrico peptide repeat) motifs. TPRs are degenerate 34 amino acid repeats that occur in tandem arrays and are involved in a variety of protein-protein interactions. TPR motifs are often found in scaffolding proteins in large complexes. The nine TPRs, each forming two antiparallel alpha helices, together create a nine alpha superhelical structure. This structure creates a groove in the TPR motifs that binds the C terminal of the third TPR helix (Lapouge et al, 2000).
Please Reload Image.
The p67phox TPR segment also contains a beta hairpin < >created by a 20 amino acid insert between the third and fourth TPR repeats. The beta hairpin is positioned by hydrogen bonds with His-69, Val-72, Glu-96, Leu-98, Ile-99, Glu-122, Val-123, and Leu-124. < > Positioning of the beta hairpin also relies on hydrogen bonds between Gly-113 and Arg-184 < > and between Glu-115andLys-181.< >
The p67phox regions necessary for Rac1 binding are contained within the N-terminus of p67phox (Lapouge et al). These binding interactions are localized to the p67phox TPR region beta hairpin structure and loops connecting TPR1 to TPR2, and TPR2 to TPR3. These contacts include both direct and water-mediated hydrogen bonds (lapouge et al, 2000).
The beta hairpin insertion Asp-108p67contactsThr-25 of Rac1.< > Arg-102p67hydrogen bonds to Ala-159, Leu-160, Asn-26,and Ser-22 of Rac1. < > Asn-104p67hydrogen bonds to Asn-26Rac1andGln-162Rac1< >.
The N terminus and C terminus of Rac1 are localized to the same area of the protein's 3D structure. Rac1 interactions with p67phox are localized to this region of the N and C termini, including Helix 1, the N terminal of Switch I, and the loop connecting beta chain 5 to alpha chain 5.
These contacts include hydrogen bonds between Gly-30Rac1andAsp-67p67 < >and between Glu-31Rac1andSer-37p67< >. No contacts are made between p67phox and Rac Switch II or the insertion helix (Lapouge et al, 2000).
Park, JB. Phagocytosis induces superoxide formation and apoptosis in macrophages. 2003. Experimental and Molecular Medicine, 35 (5): 325-335.
Bishop, A., A. Hall. Rho GTPases and their effector proteins. 2000. Biochemical Journal, 348; 241-255.
Diekmann, D., A. Abo, C. Johnston, A. Segal, A. Hall. Interactions of Rac with p67phox and regulation of phagocytic NADPH oxidase acitivity. 1994. Science, 265: 531-533.
Abo, A., E. Pci, A. Hall, N. Totty, C. Teahan, A. Segal. Activation of the NADPH oxidase involved the small GTP-binding protein p21Rac1. 1991. Nature, 353: 668-670.
Leusen J., A. de Klein, P. Hilarius, A. Ahlin, J. Palmblad, C. Smith, D. Diekmann, A. Hall, A. Vanhoeven, D. Roos. Disturbed interaction of p21-rac with mutated p67-phox causes chronic granulomatous disease. 1996. Journal of Experimental Medicine, 184: 1243-1249.
*Special thanks to Gabe Schine for help with formating.*