| Domain/Region | Structure/Composition | Function | Conservation |
|---|---|---|---|
| Domain I (G-domain) | N-terminal GTPase domain with a central 6-stranded β-sheet surrounded by 5 α-helices; contains the GTP/GDP-binding site and the canonical mobile elements of translational GTPases (switch I, switch II, and P-loop) (pqac-00000005, pqac-00000006) | Binds and hydrolyzes guanine nucleotides; mediates ribosome-dependent GTPase activity required for EF4/GUF1 action on translating ribosomes and conformational cycling during translation quality control/back-translocation-like remodeling (pqac-00000005, pqac-00000007, pqac-00000011) | Highly conserved across EF-G/EF4/BipA-like translational GTPases and across bacteria and organellar homologs, including mitochondrial GUF1 (pqac-00000003, pqac-00000005, pqac-00000011) |
| Domain II | Signature twisted β-barrel motif shared among translational GTPases (pqac-00000006) | Contributes to the conserved ribosome-binding architecture of EF4 and helps position the factor on the ribosome during elongation-state surveillance (pqac-00000005, pqac-00000007) | Conserved and topologically equivalent among EF-G, EF4, and BipA family members (pqac-00000006) |
| Domain III | α/β domain containing a 4-stranded β-sheet flanked by 2 α-helices; in EF4 its orientation differs from EF-G, contributing to distinct global conformation (pqac-00000006) | Helps shape the overall factor conformation and thereby supports EF4-specific engagement with ribosome states linked to reverse translocation and translation control (pqac-00000006, pqac-00000007) | Conserved fold shared with EF-G and BipA, though relative orientation varies and likely underlies functional divergence (pqac-00000006) |
| Domain V | α/β domain with a 4-stranded β-sheet flanked by 2 α-helices; directly contacts the G-domain in EF4, unlike the rotated arrangement seen in BipA (pqac-00000006) | Participates in the common translational GTPase scaffold and supports ribosome interaction and factor conformational organization during translation (pqac-00000005, pqac-00000006) | Conserved topologically among EF-G/EF4/BipA-like factors (pqac-00000006) |
| C-terminal domain (CTD) | EF4-specific domain replacing EF-G domain IV; comprises one long α-helix cradled by four short β-strands and occupies a distinct position from BipA CTD and EF-G domain IV (pqac-00000006) | Makes extensive contacts with ribosome-bound tRNAs; structural studies indicate interaction with acceptor stems of A-site and P-site tRNAs and support EF4/GUF1 roles in back-translocation-like remodeling and stabilization of specific ribosome conformations (pqac-00000003, pqac-00000006, pqac-00000007) | Characteristic of EF4/GUF1 family and absent from canonical EF-G, helping define EF4-specific function despite the shared core with other translational GTPases (pqac-00000005, pqac-00000006) |
| G1 box / GKS motif | Conserved nucleotide-binding sequence motif within the G-domain; in related mitochondrial translation GTPases, the G1 box follows the GX2NXGK(S/T) consensus, and the GKS residues contact the α- and β-phosphates of GDP/GTP (pqac-00000008) | Essential for guanine nucleotide binding and therefore for in vivo function; mutational disruption of the GKS motif abolishes translation-associated activity in mitochondrial ribosome-associated GTPases, supporting the same mechanistic requirement for GUF1/EF4 family members (pqac-00000008) | Strongly conserved across TRAFAC-family ribosome-associated GTPases and consistent with UniProt domain assignment for A0A2U1PS28 as a translation-factor GTPase (pqac-00000005, pqac-00000008) |
| Switch regions | Conserved mobile elements of the G-domain, including switch I and switch II, typical of translational GTPases (pqac-00000006) | Transmit nucleotide state to conformational changes that regulate ribosome engagement, GTP hydrolysis, and release; central to coupling guanine nucleotide state with EF4/GUF1 action on the ribosome (pqac-00000005, pqac-00000007) | Broadly conserved among translational GTPases, including EF-G, EF4, and organellar GUF1 homologs (pqac-00000005, pqac-00000006) |
| P-loop | Conserved phosphate-binding loop within the G-domain nucleotide-binding pocket (pqac-00000006) | Coordinates phosphate groups of bound GDP/GTP and is fundamental to ribosome-dependent GTPase activity during translation factor cycling (pqac-00000005, pqac-00000007) | Universally conserved hallmark of Ras-like/TRAFAC GTPases and retained in EF4/GUF1 family proteins (pqac-00000005, pqac-00000006) |


*Table: This table summarizes the conserved structural architecture of GUF1/EF4 and links individual domains and motifs to their known or inferred roles in ribosome-dependent GTPase activity and mitochondrial translation. It is useful for functional annotation of the Artemisia annua GUF1 homolog by separating well-supported structural features from mechanistic inference.*