Structure of G Domain among G Proteins

Structure of G Domain among G Proteins: History

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Contributor:

Zhaoheng Lin

Rongfang Li

Zhiwei Han

Yi Liu

Liyang Gao

Suchang Huang

Ying Miao

Rui Miao

The ancient guanine nucleotide-binding (G) proteins are a group of critical regulatory and signal transduction proteins, widely involved in diverse cellular processes of all kingdoms of life. YchF is a kind of universally conserved novel unconventional G protein that appears to be crucial for growth and stress response in eukaryotes and bacteria. YchF is able to bind and hydrolyze both adenine nucleoside triphosphate (ATP) and guanosine nucleoside triphosphate (GTP), unlike other members of the P-loop GTPases.

YchF
growth
stress response
P-loop NTPase

1. Structural Characterization of G Domain of G Proteins

All G proteins utilize the G domain to bind and hydrolyze nucleotides, which contains five structurally conserved motifs (G boxes): G1 motif (G1 box) adopting the sequence pattern GxxxxGK(S/T), G2 motif (G2 box) adopting the sequence pattern x(T/S)x, G3 motif (G3 box) adopting the sequence pattern hhhDxxG, G4 motif (G4 box) adopting the sequence pattern (N/T)KxD and G5 motif (G5 box) adopting the sequence pattern (T/G)(C/S)A ^[1] (Figure 1A). The so-called P-loop or walk A motif is the G1 box that binds to α- and β-phosphate of nucleotides. The walk B motif consists of a G2 box and a G3 box that anchor to the terminal γ-phosphate of nucleotide. The G3 box has a conserved aspartic acid (Asp/D) residue in contact with the co-factor magnesium (Mg²⁺), which is crucial for nucleotide binding and hydrolysis (Figure 1A). In addition, the walk B motif overlapping with the switch I and switch II regions undergo a conformational change accompanied by nucleotide hydrolysis, which governs effector binding. The G4 box determines the guanosine or adenosine signature, and the G5 box supports specific recognition.

Figure 1. Structural characterization of the evolutionarily conserved unconventional G protein YchF. (A) Schematic representation of the structure of universally conserved unconventional G protein YchF. (B) Structural alignment of the nontypical G4 motif (N/T)(M/L/V)xE in the YchF subfamily (E. coli YchF is green, hOLA1 is cyan, S. pombe YchF is yellow, OsYchF1 is brown, T. thermophilus YchF is red). The amino acid residues are shown as sticks. (C) Electron density surface of the apo-structure of OsYchF1 (Protein Data Bank (PDB) code: 5EE0). Negatively charged amino acid residues are red, and positively charged amino acid residues are blue.

2. Structural Characterization of G Domain of YchF

The G domain of universally conserved unconventional G protein YchF maintains five fingerprint motifs as other G proteins. The G1, G2, G3, and G5 boxes are invariant with other G proteins, but the G4 box in the YchF subfamily shows a nontypical (N/T)(M/L/V)xE amino acid sequence instead of (N/T)KxD (Figure 1A,B; Table 1). Thus, the members in the YchF subfamily are capable of binding and hydrolyzing both adenine nucleoside triphosphate (ATP) and guanosine nucleoside triphosphate (GTP) ^[2].

Table 1. Summary of function-related amino acid residues in YchF subfamily.

Homolog	Species	Residues	Location	Supportive Reasons/Effects	Functions	References
E. coli YchF	Escherichia coli	His114	A highly flexible loop of G domain	Supporting the flexible loop to reach a catalytically active conformation	Critical for ATPase activity (+)	^[3]
E. coli YchF	Escherichia coli	Cys35	G2 motif	Allows YchF dimerization via a disulfide bridge	Critical for ATPase activity (−)	^[4]
E. coli YchF	Escherichia coli	Lys78 (Arg)	G domain	YchF-K78A mutant shows similar hydrolysis activities in presence of Na⁺ or K⁺, but K78R mutant retained potassium specific stimulation of ATPase activity	Plays a key role in determining the potassium dependent ATPase activity	^[5]
hOLA1	Homo sapiens	Leu96	G domain (next to G3 motif)	A conserved Gln residue involved in GTP hydrolysis in Ras-like GTPases has been replaced	Inactivates Ras-like GTPases	^[6]
E. coli YchF	Escherichia coli	Ser16 (Ser36 in H. sapiens)	G1 motif	Ser16 phosphorylated when H₂O₂ absence; Dissociation of KatG	Supports the ATPase activity; Detoxifies H₂O₂	^[6]^[7]
E. coli YchF	Escherichia coli	Leu76	G3 motif	Hallmark for HAS-NTPase	Slightly affects ATPase activity (+)	^[3]
hOLA1	Homo sapiens	Thr37	G domain	The main chain amide of Thr37 contacts the α-phosphate of AMPPCP	Supports the ATPase activity	^[6]
hOLA1	Homo sapiens	Ser36/Val33	G1 motif	The main chain amide of Ser36 and Val33 contacts the β-phosphate of AMPPCP	Supports the ATPase activity	^[6]
hOLA1	Homo sapiens	Asn32	G1 motif	The main chain amide of Asn32 forms a hydrogen bond to the γ-phosphate of AMPPCP	Supports the ATPase activity	^[6]
hOLA1	Homo sapiens	Asn230	G4 motif	Its mutation to alanine abolished nucleotide binding	Contribute to nucleotide binding	^[6]
hOLA1	Homo sapiens	Leu231	G4 motif	Specificity for adenine binding is based on the interaction between the adenine N-6 group and Leu231 main chain CO in G4 motif	Make YchF preference for ATP rather than GTP	^[6]
hOLA1	Homo sapiens	Ser310	TGS domain	The H-bond between Ser310 O_γ and the exocyclic N-6 of an adenine is formed in a position similar to the ppGpp O-6	Make YchF preference for ATP rather than GTP	^[6]
hOLA1	Homo sapiens	Phe127	Coiled-coil domain	Mutating this residue to Ala diminishes ATP binding drastically	Contribute to base recognition	^[6]
AtYchF1	Arabidopsis thaliana	Glu345	TGS domain	Conserved and solvent-exposed	Most critical for its interaction with the regulator, GAP1	^[8]

Amino acids in brackets indicate that there are other known residues presenting at the same position in the orthologs; In the “functions” column, (+) means upregulation, and (−) means down-regulation.

3. Structural Comparison of G Domains among Selected YchF, Small G Protein, and Heterotrimeric G Protein α-Subunit

Herein, a heterotrimeric G protein α-subunit in the rat (Rattus norvegicus) and a human (Homo Sapien) small G protein Ras-related G protein C was chosen to compare with OsYchF1, a rice (Oryza sativa) ortholog of YchF in plants. In contrast to R. norvegicus heterotrimeric G protein α-subunit and human Ras-related G protein C, the novel G4 motif and G5 motif of OsYchF1 support either ATP or GTP binding in the nucleotide-binding site of OsYchF1 (Figure 1B and Figure 2A). According to the crystal structure of OsYchF1 in the presence of the ATP non-hydrolyzed homolog AMPPNP (Protein Data Bank (PDB) code: 5EE3), the backbone carboxyl group of methionine (M231) in the G4 motif of OsYchF1 forms a hydrogen bond with the adenine 6-amino group of AMPPNP (Figure 2A) ^[2]^[9]. This allows for the non-hydrolytic AMPPNP to be able to fit into the OsYchF1 nucleotide-binding site (Figure 2A). The structural alignments of OsYchF1 with R. norvegicus heterotrimeric G protein α-subunit (PDB code: 1SVS) and human Ras-related G protein C (PDB code: 3LLU) revealed that the side chain of asparagine (Asn) in the G4 motif of OsYchF1 could not turn back and interact with the 2-amino group of guanosine, unlike the other two proteins. However, the crystal structure of OsYchF1 in the presence of GppNHp (PDB code: 5EE9), a non-hydrolyzed homolog of GTP, showed that the G5 motif of OsYchF1 can form a hydrogen bond with the guanosine base group of GppNHp. This finding partially explains why OsYchF1 is capable of binding to GTP as well (Figure 2B) ^[2]^[9]. Moreover, the G1 motif (P-loop) is highly conserved and consistent among OsYchF1, R. norvegicus heterotrimeric G protein α-subunit, and human Ras-related G protein C (Figure 2A,B). The G1 motif of OsYchF1 interacts with the triphosphate of nucleotides, resembling the G1 motif of R. norvegicus heterotrimeric G protein α-subunit and human Ras-related Protein C (Figure 2A,B). In the OsYchF1 G4 motif mutant, however, methionine (Met) and glutamine (Glu) were replaced by lysine (Lys) and aspartic acid (Asp), respectively. With this change in amino acids, OsYchF1 obtained GTP priority again, indicating that the OsYchF1 G4 motif indeed determines ATP or GTP recognition ^[2]^[9].

Figure 2. Structural alignments of OsYchF1 hOLA1 H. sapiens Ras-related G protein C and R. norvegicus heterotrimeric G protein α-subunit nucleotide-binding sites in the complex with nucleotides. (A) Structural alignments of OsYchF1 (PDB code: 5EE3), hOLA1 (PDB code: 2OHF), human Ras-related G protein C (HsRas C) (PDB code: 3LLU), and R. norvegicus heterotrimeric G protein α-subunit (RnHetero) (PDB code: 1SVS) nucleotide-binding site in the complex with the ATP non-hydrolyzed homolog AMPPNP. (B) Structural alignments of OsYchF1 (PDB code: 5EE9), hOLA1, HsRas C, and RnHetero nucleotide-binding site in the complex with the GTP non-hydrolyzed homolog GppNHp. AMPPNP, GppNHp, M-231, L-231, K-138, and K-277 are shown as sticks. The G1 motif (P-loop), G4 motif, and G5 motif are shown as cartoons (OsYchF1 is green, hOLA1 is yellow, human Ras-related G protein C is cyan, and R. norvegicus heterotrimeric G protein α-subunit is pink).

This entry is adapted from the peer-reviewed paper 10.3390/life13041058

References

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