The peptide-substrate-binding domain of collagen prolyl 4-hydroxylases is a tetratricopeptide repeat domain with functional aromatic residues

Collagen prolyl 4-hydroxylases catalyze the formation of 4-hydroxyproline in -X-Pro-Gly-sequences and have an essential role in collagen synthesis. The vertebrate enzymes are alpha2beta2 tetramers in which the catalytic alpha-subunits contain separate peptide-substrate-binding and catalytic domains. We report on the crystal structure of the peptide-substrate-binding domain of the human type I enzyme refined at 2.3 A resolution. It was found to belong to a family of tetratricopeptide repeat domains that are involved in many protein-protein interactions and consist of five alpha-helices forming two tetratricopeptide repeat motifs plus the solvating helix. A prominent feature of its concave surface is a deep groove lined by tyrosines, a putative binding site for proline-rich Tripeptides. Solvent-exposed side chains of three of the tyrosines have a repeat distance similar to that of a poly-L-proline type II helix. The aromatic surface ends at one of the tyrosines, where the groove curves almost 90 degrees away from the linear arrangement of the three tyrosine side chains, possibly inducing a bent conformation in the bound peptide. This finding is consistent with previous suggestions by others that a minimal structural requirement for proline 4-hydroxylation may be a sequence in the poly-L-proline type II conformation followed by a beta-turn in the Pro-Gly segment. Site-directed mutagenesis indicated that none of the tyrosines was critical for tetramer assembly, whereas most of them were critical for the binding of a peptide substrate and inhibitor both to the domain and the alpha2beta2 enzyme tetramer.     

Structural analysis of multifunctional peptide motifs in human bifunctional tRNA synthetase: identification of RNA-binding residues and functional implications for tandem repeats

Human bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) contains three tandem repeats linking the two catalytic domains. These repeated motifs have been shown to be involved in protein-protein and protein-nucleic acid interactions. The single copy of the homologous motifs has also been found in several different aminoacyl-tRNA synthetases. The solution structure of repeat 1 (EPRS-R1) and the secondary structure of the whole appended domain containing three repeated motifs in EPRS (EPRS-R123) was determined by nuclear magnetic resonance (NMR) spectroscopy. EPRS-R1 consists of two helices (residues 679-699 and 702-721) arranged in a helix-turn-helix, which is similar to other RNA binding proteins and the j-domain of DnaJ, and EPRS-R123 is composed of three helix-turn-helix motifs linked by an unstructured loop. When tRNA is bound to the appended domain, chemical shifts of several residues in each repeat are perturbed. However, the perturbed residues in each repeat are not the same although they are in the same binding surface, suggesting that each repeat in the appended domain is dynamically arranged to maximize contacts with tRNA. The affinity of tRNA to the three-repeated motif was much higher than to the single motif. These results indicate that each of the repeated motifs has a weak intrinsic affinity for tRNA, but the repetition of the motifs may be required to enhance binding affinity. Thus, the results of this work gave information on the RNA-binding mode of the multifunctional peptide motif attached to different ARSs and the functional reason for the repetition of this motif.     

Divergence in the plasminogen-binding group a streptococcal M protein family: functional conservation of binding site and potential role for immune selection of variants

Group A streptococci (GAS) display receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M protein (PAM). Characterization of PAM genes from 12 GAS isolates showed significant variation within the plasminogen-binding repeat motifs (a1/a2) of this protein. To determine the impact of sequence variation on protein function, recombinant proteins representing five naturally occurring variants of PAM, together with a recombinant M1 protein, were expressed and purified. Equilibrium dissociation constants for the interaction of PAM variants with biotinylated Glu-plasminogen ranged from 1.58 to 4.99 nm. Effective concentrations of prototype PAM required for 50% inhibition of plasminogen binding to immobilized PAM variants ranged from 0.68 to 22.06 nm. These results suggest that although variation in the a1/a2 region of the PAM protein does affect the comparative affinity of PAM variants, the functional capacity to bind plasminogen is conserved. Additionally, a potential role for the a1 region of PAM in eliciting a protective immune response was investigated by using a mouse model for GAS infection. The a1 region of PAM was found to protect immunized mice challenged with a PAM-positive GAS strain. These data suggest a link between selective immune pressure against the plasminogen-binding repeats and the functional conservation of the binding domain in PAM variants.     

Domain interactions between streptokinase and human plasminogen.

Plasmin (Pm), the main fibrinolytic protease in the plasma, is derived from its zymogen plasminogen (Plg) by cleavage of a peptide bond at Arg(561)-Val(562). Streptokinase (SK), a widely used thrombolytic agent, is an efficient activator of human Plg. Both are multiple-domain proteins that form a tight 1:1 complex. The Plg moiety gains catalytic activity, without peptide bond cleavage, allowing the complex to activate other Plg molecules to Pm by conventional proteolysis. We report here studies on the interactions between individual domains of the two proteins and their roles in Plg activation. Individually, all three SK domains activated native Plg. While the SK alpha domain was the most active, its activity was uniquely dependent on the presence of Pm. The SK gamma domain also induced the formation of an active site in Plg(R561A), a mutant that resists proteolytic activation. The alpha and gamma domains together yielded synergistic activity, both in Plg activation and in Plg(R561A) active site formation. However, the synergistic activity of the latter was dependent on the correct N-terminal isoleucine in the alpha domain. Binding studies using surface plasmon resonance indicated that all three domains of SK interact with the Plg catalytic domain and that the beta domain additionally interacts with Plg kringle 5. These results suggest mechanistic steps in SK-mediated Plg activation. In the case of free Plg, complex formation is initiated by the rapid and obligatory interaction between the SK beta domain and Plg kringle 5. After binding of all SK domains to the catalytic domain of Plg, the SK alpha and gamma domains cooperatively induce the formation of an active site within the Plg moiety of the activator complex. Substrate Plg is then recognized by the activator complex through interactions predominately mediated by the SK alpha domain.     

Biological role of the two overlapping poly(A)-binding protein interacting motifs 2 (PAM2) of eukaryotic releasing factor eRF3 in mRNA decay

Eukaryotic releasing factor GSPT/eRF3 mediates translation termination-coupled mRNA decay via interaction with a cytosolic poly(A)-binding protein (PABPC1). A region of eRF3 containing two overlapping PAM2 (PABPC1-interacting motif 2) motifs is assumed to bind to the PABC domain of PABPC1, on the poly(A) tail of mRNA. PAM2 motifs are also found in the major deadenylases Caf1–Ccr4 and Pan2–Pan3, whose activities are enhanced upon PABPC1 binding to these motifs. Their deadenylase activities are regulated by eRF3, in which two overlapping PAM2 motifs competitively prevent interaction with PABPC1. However, it is unclear how these overlapping motifs recognize PABC and regulate deadenylase activity in a translation termination-coupled manner. We used a dominant-negative approach to demonstrate that the N-terminal PAM2 motif is critical for eRF3 binding to PABPC1 and that both motifs are required for function. Isothermal titration calorimetry (ITC) and NMR analyses revealed that the interaction is in equilibrium between the two PAM2–PABC complexes, where only one of the two overlapping PAM2 motifs is PABC-bound and the other is PABC-unbound and partially accessible to the other PABC. Based on these results, we proposed a biological role for the overlapping PAM2 motifs in the regulation of deadenylase accessibility to PABPC1 at the 3′ end of poly(A).     

A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly

Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1-7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNDeltaIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9-10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4-7, had no effect on assembly. In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.     

Structure of a thrombospondin C-terminal fragment reveals a novel calcium core in the type 3 repeats

Thrombospondins (TSPs) are extracellular regulators of cell-matrix interactions and cell phenotype. The most highly conserved region of all TSPs are the calcium-binding type 3 (T3) repeats and the C-terminal globular domain (CTD). The crystal structure of a cell-binding TSP-1 fragment, spanning three T3 repeats and the CTD, reveals a compact assembly. The T3 repeats lack secondary structure and are organised around a core of calcium ions; two DxDxDGxxDxxD motifs per repeat each encapsulate two calcium ions in a novel arrangement. The CTD forms a lectin-like beta-sandwich and contains four strictly conserved calcium-binding sites. Disruption of the hairpin structure of T3 repeats 6 and 7 decreases protein secretion and stability. The availability for cell attachment of an RGD motif in T3 repeat 7 is modulated by calcium loading. The central architectural role of calcium explains how it is critical for the functions of the TSP C-terminal region. Mutations in the T3 repeats of TSP-5/COMP, which cause two human skeletal disorders, are predicted to disrupt the tertiary structure of the T3-CTD assembly.     

Analysis of the interactions between streptokinase domains and human plasminogen.

The contrasting roles of streptokinase (SK) domains in binding human Glu1-plasminogen (Plg) have been studied using a set of proteolytic fragments, each of which encompasses one or more of SK's three structural domains (A, B, C). Direct binding experiments have been performed using gel filtration chromatography and surface plasmon resonance. The latter technique has allowed estimation of association and dissociation rate constants for interactions between Plg and intact SK or SK fragments. Each of the SK fragments that contains domain B (fragments A2-B-C, A2-B, B-C, and B) binds Plg with similar affinity, at a level approximately 100- to 1,000-fold lower than intact SK. Experiments using 10 mM 6-aminohexanoic acid or 50 mM benzamidine demonstrate that either of these two lysine analogues abolishes interaction of domain B with Plg. Isolated domain C does not show detectable binding to Plg. Moreover, the additional presence of domain C within other SK fragments (B-C and A2-B-C) does not alter significantly their affinities for Plg. In addition, Plg-binding by a noncovalent complex of two SK fragments that contains domains A and B is similar to that of domain B. By contrast, species containing domain B and both domains A and C (intact SK and the two-chain complex A1 x A2-B-C) show a significantly higher affinity for Plg, which could not be completely inhibited by saturating amounts of 6-AHA. These results show that SK domain B interacts with Plg in a lysine-dependent manner and that although domains A and C do not appear independently to possess affinity for Plg, they function cooperatively to establish the additional interactions with Plg to form an efficient native-like Plg activator complex.     

Paip1 interacts with poly(A) binding protein through two independent binding motifs

The 3' poly(A) tail of eukaryotic mRNAs plays an important role in the regulation of translation. The poly(A) binding protein (PABP) interacts with eukaryotic initiation factor 4G (eIF4G), a component of the eIF4F complex, which binds to the 5' cap structure. The PABP-eIF4G interaction brings about the circularization of the mRNA by joining its 5' and 3' termini, thereby stimulating mRNA translation. The activity of PABP is regulated by two interacting proteins, Paip1 and Paip2. To study the mechanism of the Paip1-PABP interaction, far-Western, glutathione S-transferase pull-down, and surface plasmon resonance experiments were performed. Paip1 contains two binding sites for PABP, PAM1 and PAM2 (for PABP-interacting motifs 1 and 2). PAM2 consists of a 15-amino-acid stretch residing in the N terminus, and PAM1 encompasses a larger C-terminal acidic-amino-acid-rich region. PABP also contains two Paip1 binding sites, one located in RNA recognition motifs 1 and 2 and the other located in the C-terminal domain. Paip1 binds to PABP with a 1:1 stoichiometry and an apparent K(d) of 1.9 nM.     

Dissecting mannose 6-phosphate-insulin-like growth factor 2 receptor complexes that control activation and uptake of plasminogen in cells

The plasminogen (Plg) activation cascade on the cell surface plays a central role in cell migration and is involved in a plethora of physiological and pathological processes. Its regulation is coordinated by many receptors, in particular the urokinase-type plasminogen activator receptor (uPAR, CD87), receptors that physically interact and functionally cooperate with uPAR, and Plg binding molecules. Here we studied the impact of one of the Plg binding molecules, the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P-IGF2R, CD222), on cellular Plg activation. By developing both in vitro and in vivo Plg activation assays on size-fractionated lysates of M6P-IGF2R-silenced cells, we identified Plg-associated complexes with M6P-IGF2R as the regulatory factor. Using lipid raft preserving versus dissolving detergents, we found lipid dependence of the Plg regulatory function of these complexes. Furthermore, M6P-IGF2R-silencing in uPAR-positive human cell lines reduced internalization of Plg, resulting in elevated Plg activation. In contrast, the expression of human M6P-IGF2R in mouse embryonic fibroblasts derived from M6P-IGF2R knock-out mice enhanced Plg internalization. Finally, peptide 18-36 derived from the Plg-binding site within M6P-IGF2R enhanced Plg uptake. Thus, by targeting Plg to endocytic pathways, M6P-IGF2R appears to control Plg activation within cells that might be important to restrict plasmin activity to specific sites and substrates.     

The maintenance of high affinity plasminogen binding by group A streptococcal plasminogen-binding M-like protein is mediated by arginine and histidine residues within the a1 and a2 repeat domains

Subversion of the plasminogen activation system is implicated in the virulence of group A streptococci (GAS). GAS displays receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M-like protein (PAM). The plasminogen binding domain of PAM is highly variable, and this variation has been linked to host selective immune pressure. Site-directed mutagenesis of full-length PAM protein from an invasive GAS isolate was undertaken to assess the contribution of residues in the a1 and a2 repeat domains to plasminogen binding function. Mutagenesis to alanine of key plasminogen binding lysine residues in the a1 and a2 repeats (Lys98 and Lys111) did not abrogate plasminogen binding by PAM nor did additional mutagenesis of Arg101 and His102 and Glu104, which have previously been implicated in plasminogen binding. Plasminogen binding was only abolished with the additional mutagenesis of Arg114 and His115 to alanine. Furthermore, mutagenesis of both arginine (Arg101 and Arg114) and histidine (His102 and His115) residues abolished interaction with plasminogen despite the presence of Lys98 and Lys111 in the binding repeats. This study shows for the first time that residues Arg101, Arg114, His102, and His115 in both the a1 and a2 repeat domains of PAM can mediate high affinity plasminogen binding. These data suggest that highly conserved arginine and histidine residues may compensate for variation elsewhere in the a1 and a2 plasminogen binding repeats, and may explain the maintenance of high affinity plasminogen binding by naturally occurring variants of PAM.     

Heterochromatin Protein 1a (HP1a) Partner Specificity Is Determined by Critical Amino Acids in the Chromo Shadow Domain and C-terminal Extension

Drosophila melanogaster Heterochromatin Protein 1a (HP1a) is an essential protein critical for heterochromatin assembly and regulation. Its chromo shadow domain (CSD) homodimerizes, a requirement for binding protein partners that contain a PXVXL motif. How does HP1a select among its many different PXVXL-containing partners? HP1a binds tightly to Heterochromatin Protein 2 (HP2), but weakly to PIWI. We investigated differences in homodimerization and the impact of the C-terminal extension (CTE) by contrasting HP1a to its paralogue, HP1b. HP1a and HP1b differ in the dimerization interface, with HP1a having an Arg at position 188 rather than Glu. We find that while this substitution reduces the dimerization constant, it does not impact the binding surface as demonstrated by unchanged partner binding affinities. However, the CTE (only 4 residues in HP1a as compared with 87 residues in HP1b) is critical; the charged residues in HP1a are necessary for tight peptide binding. Examining a panel of amino acid substitutions in the HP1a CSD, we find that Leu-165 in HP1a interacts with HP2 but not PIWI, supporting the conclusion that different sites in the binding surface provide discrimination for partner selection. Partner sequence is also critical for affinity, as the remaining difference in binding between HP2 and PIWI polypeptides is eliminated by swapping the PXVXL motifs between the two. Taken together, these studies indicate that the binding surface of the HP1a CSD plus its short CTE provide the needed discrimination among HP1a''s partners, and that the CTE is important for differentiating the interactions of the Drosophila HP1 paralogs.     

Structural analysis of the interactions between paxillin LD motifs and alpha-parvin

The adaptor protein paxillin contains five conserved leucine-rich (LD) motifs that interact with a variety of focal adhesion proteins, such as alpha-parvin. Here, we report the first crystal structure of the C-terminal calponin homology domain (CH(C)) of alpha-parvin at 1.05 A resolution and show that it is able to bind all the LD motifs, with some selectivity for LD1, LD2, and LD4. Cocrystal structures with these LD motifs reveal the molecular details of their interactions with a common binding site on alpha-parvin-CH(C), which is located at the rim of the canonical fold and includes part of the inter-CH domain linker. Surprisingly, this binding site can accommodate LD motifs in two antiparallel orientations. Taken together, these results reveal an unusual degree of binding degeneracy in the paxillin/alpha-parvin system that may facilitate the assembly of dynamic signaling complexes in the cell.     

The highly repetitive region of the Helicobacter pylori CagY protein comprises tandem arrays of an alpha-helical repeat module

The cag-pathogenicity-island-encoded type IV secretion system of Helicobacter pylori functions to translocate the effector protein CagA directly through the plasma membrane of gastric epithelial cells. Similar to other secretion systems, the Cag type IV secretion system elaborates a surface filament structure, which is unusually sheathed by the large cag-pathogenicity-island-encoded protein CagY. CagY is distinguished by unusual amino acid composition and extensive repetitive sequence organised into two defined repeat regions. The second and major repeat region (CagY_rpt2) has a regular disposition of six repetitive motifs, which are subject to deletion and duplication, facilitating the generation of CagY size and phenotypic variants. In this study, we show CagY_rpt2 to comprise two highly thermostable and acid-stable α-helical structural motifs, the most abundant of which (motif A) occurs in tandem arrays of one to six repeats terminally flanked by single copies of the second repeat (motif B). Isolated motifs demonstrate hetero- and homomeric interactions, suggesting a propensity for uniform assembly of discrete structural subunit motifs within the larger CagY_rpt2 structure. Consistent with this, CagY proteins comprising substantially different repeat 2 motif organisations demonstrate equivalent CagA translocation competence, illustrating a remarkable structural and functional tolerance for precise deletion and duplication of motif subunits. We provide the first insight into the structural basis for CagY_rpt2 assembly that accommodates both the variable motif sequence composition and the extensive contraction/expansion of repeat modules within the CagY_rpt2 region.     

Structural basis of ligand recognition by PABC, a highly specific peptide-binding domain found in poly(A)-binding protein and a HECT ubiquitin ligase

The C-terminal domain of poly(A)-binding protein (PABC) is a peptide-binding domain found in poly(A)-binding proteins (PABPs) and a HECT (homologous to E6-AP C-terminus) family E3 ubiquitin ligase. In protein synthesis, the PABC domain of PABP functions to recruit several translation factors possessing the PABP-interacting motif 2 (PAM2) to the mRNA poly(A) tail. We have determined the solution structure of the human PABC domain in complex with two peptides from PABP-interacting protein-1 (Paip1) and Paip2. The structures show a novel mode of peptide recognition, in which the peptide binds as a pair of beta-turns with extensive hydrophobic, electrostatic and aromatic stacking interactions. Mutagenesis of PABC and peptide residues was used to identify key protein-peptide interactions and quantified by isothermal calorimetry, surface plasmon resonance and GST pull-down assays. The results provide insight into the specificity of PABC in mediating PABP-protein interactions.     

The MLLE Domain of the Ubiquitin Ligase UBR5 Binds to Its Catalytic Domain to Regulate Substrate Binding

E3 ubiquitin ligases catalyze the transfer of ubiquitin from an E2-conjugating enzyme to a substrate. UBR5, homologous to the E6AP C terminus (HECT)-type E3 ligase, mediates the ubiquitination of proteins involved in translation regulation, DNA damage response, and gluconeogenesis. In addition, UBR5 functions in a ligase-independent manner by prompting protein/protein interactions without ubiquitination of the binding partner. Despite recent functional studies, the mechanisms involved in substrate recognition and selective ubiquitination of its binding partners remain elusive. The C terminus of UBR5 harbors the HECT catalytic domain and an adjacent MLLE domain. MLLE domains mediate protein/protein interactions through the binding of a conserved peptide motif, termed PAM2. Here, we characterize the binding properties of the UBR5 MLLE domain to PAM2 peptides from Paip1 and GW182. The crystal structure with a Paip1 PAM2 peptide reveals the network of hydrophobic and ionic interactions that drive binding. In addition, we identify a novel interaction of the MLLE domain with the adjacent HECT domain mediated by a PAM2-like sequence. Our results confirm the role of the MLLE domain of UBR5 in substrate recruitment and suggest a potential role in regulating UBR5 ligase activity.     

Role of the stable signal peptide and cytoplasmic domain of G2 in regulating intracellular transport of the Junín virus envelope glycoprotein complex

Enveloped viruses utilize the membranous compartments of the host cell for the assembly and budding of new virion particles. In this report, we have investigated the biogenesis and trafficking of the envelope glycoprotein (GP-C) of the Junín arenavirus. The mature GP-C complex is unusual in that it retains a stable signal peptide (SSP) as an essential component in association with the typical receptor-binding (G1) and transmembrane fusion (G2) subunits. We demonstrate that, in the absence of SSP, the G1-G2 precursor is restricted to the endoplasmic reticulum (ER). This constraint is relieved by coexpression of SSP in trans, allowing transit of the assembled GP-C complex through the Golgi and to the cell surface, the site of arenavirus budding. Transport of a chimeric CD4 glycoprotein bearing the transmembrane and cytoplasmic domains of G2 is similarly regulated by SSP association. Truncations to the cytoplasmic domain of G2 abrogate SSP association yet now permit transport of the G1-G2 precursor to the cell surface. Thus, the cytoplasmic domain of G2 is an important determinant for both ER localization and its control through SSP binding. Alanine mutations to either of two dibasic amino acid motifs in the G2 cytoplasmic domain can also mobilize the G1-G2 precursor for transit through the Golgi. Taken together, our results suggest that SSP binding masks endogenous ER localization signals in the cytoplasmic domain of G2 to ensure that only the fully assembled, tripartite GP-C complex is transported for virion assembly. This quality control process points to an important role of SSP in the structure and function of the arenavirus envelope glycoprotein.     

Simultaneous binding of two peptidyl ligands by a SRC homology 2 domain

Src homology 2 (SH2) domains mediate protein-protein interactions by recognizing phosphotyrosine (pY)-containing sequences of target proteins. In all of the SH2 domain-pY peptide interactions described to date, the SH2 domain binds to a single pY peptide. Here, determination of the cocrystal structure of the N-terminal SH2 domain of phosphatase SHP-2 bound to a class IV peptide (VIpYFVP) revealed a noncanonical 1:2 (protein-peptide) complex. The first peptide binds in a canonical manner with its pY side chain inserted in the usual binding pocket, while the second pairs up with the first to form two antiparallel β-strands that extend the central β-sheet of the SH2 domain. This unprecedented binding mode was confirmed in the solution phase by NMR experiments and shown to be adopted by pY peptides derived from cellular proteins. Site-directed mutagenesis and surface plasmon resonance studies revealed that the binding of the first peptide is pY-dependent, but phosphorylation is not required for the second peptide. Our findings suggest a potential new function for the SH2 domain as a molecular clamp to promote dimerization of signaling proteins.     

La-related protein 4 binds poly(A), interacts with the poly(A)-binding protein MLLE domain via a variant PAM2w motif, and can promote mRNA stability

The conserved RNA binding protein La recognizes UUU-3'OH on its small nuclear RNA ligands and stabilizes them against 3'-end-mediated decay. We report that newly described La-related protein 4 (LARP4) is a factor that can bind poly(A) RNA and interact with poly(A) binding protein (PABP). Yeast two-hybrid analysis and reciprocal immunoprecipitations (IPs) from HeLa cells revealed that LARP4 interacts with RACK1, a 40S ribosome- and mRNA-associated protein. LARP4 cosediments with 40S ribosome subunits and polyribosomes, and its knockdown decreases translation. Mutagenesis of the RNA binding or PABP interaction motifs decrease LARP4 association with polysomes. Several translation and mRNA metabolism-related proteins use a PAM2 sequence containing a critical invariant phenylalanine to make direct contact with the MLLE domain of PABP, and their competition for the MLLE is thought to regulate mRNA homeostasis. Unlike all ～150 previously analyzed PAM2 sequences, LARP4 contains a variant PAM2 (PAM2w) with tryptophan in place of the phenylalanine. Binding and nuclear magnetic resonance (NMR) studies have shown that a peptide representing LARP4 PAM2w interacts with the MLLE of PABP within the affinity range measured for other PAM2 motif peptides. A cocrystal of PABC bound to LARP4 PAM2w shows tryptophan in the pocket in PABC-MLLE otherwise occupied by phenylalanine. We present evidence that LARP4 expression stimulates luciferase reporter activity by promoting mRNA stability, as shown by mRNA decay analysis of luciferase and cellular mRNAs. We propose that LARP4 activity is integrated with other PAM2 protein activities by PABP as part of mRNA homeostasis.