Degeneracy and function of the ubiquitous RVXF motif that mediates binding to protein phosphatase-1

Most interactors of protein phosphatase-1 (PP1) contain a variant of a so-called "RVXF" sequence that binds to a hydrophobic groove of the catalytic subunit. A combination of sequence alignments and site-directed mutagenesis has enabled us to further define the consensus sequence for this degenerate motif as [RK]-X(0-1)-[VI]-[P]-[FW], where X denotes any residue and [P] any residue except Pro. Naturally occurring RVXF sequences differ in their affinity for PP1, and we show by swapping experiments that this binding affinity is an important determinant of the inhibitory potency of the regulators NIPP1 and inhibitor-1. Also, inhibition by NIPP1-(143-224) was retained when the RVXF motif (plus the preceding Ser) was swapped for either of two unrelated PP1-binding sequences from human inhibitor-2, i.e. KGILK or RKLHY. Conversely, the KGILK motif of inhibitor-2 could be functionally replaced by the RVXF motif of NIPP1. Our data provide additional evidence for the view that the RVXF and KGILK motifs function as anchors for PP1 and thereby promote the interaction of secondary binding sites that determine the activity and substrate specificity of the enzyme.     

Binding of manganese stabilizing protein to photosystem II: identification of essential N-terminal threonine residues and domains that prevent nonspecific binding

The N-terminus of spinach photosystem II manganese stabilizing protein (MSP) contains two amino acid sequences, (4)KRLTYD(10)E and (15)TYL(18)E, that are necessary for binding of two copies of this subunit to the enzyme [Popelkova et al. (2002) Biochemistry 41, 10038-10045]. To better understand the basis of MSP-photosystem II interactions, the role of threonine residues in the highly conserved motifs T(Y/F)DE and TY has been characterized. Deletion mutants lacking the first 5, 6, 7, and 15 amino acid residues at the N-terminus of the protein were examined for their ability to reconstitute activity in MSP-depleted photosystem II. The results reported here show that truncations of five and six amino acid residues (mutants DeltaR5M and DeltaL6M mutants) have no negative effect on recovery of oxygen evolution activity or on binding of MSP to photosystem II. Deletion of seven residues (mutant DeltaT7M) decreases reconstitution activity to 40% of the control value and reduces functional binding of the mutant protein to photosystem II from two to one copy. Deletion of 15 amino acid residues (mutant DeltaT15M) severely impairs functional binding of MSP, and lowers O(2) evolution activity to less than 20% of the control. DeltaT7M is the only mutant that exhibited neither nonspecific binding to photosystem II nor changes in tertiary structure. These, and previous results, show that the highly conserved Thr7 and Thr15 residues of MSP are required for functional binding of two copies of the eukaryotic protein to photosystem II. Although the N-terminal domains, (1)EGGKR(6)L, (8)YDEIQS(14)K, and (16)YL(18)E of spinach MSP are unnecessary for specific, functional binding interactions, these sequences are necessary to prevent nonspecific binding of the protein to photosystem II.     

Integrin beta1-chain residues involved in substrate recognition and specificity of binding to invasin

A highly conserved 14-amino-acid region of the chicken beta1 integrin chain (beta1chk residues 151-168) hypothesized to be involved in divalent cation coordination was analysed to determine whether invasin uses the same structural determinants as fibronectin (Fn) to recognize receptors. For the most part, both proteins required similar beta1 chain residues for integrin recognition, although the relative preference of the integrin for the two substrates could be inverted mutationally. Substitution mutations in the amino terminal residues of this region resulted in defective binding to both substrates by the receptor, while substitutions at the carboxyl-terminal end of this region were better tolerated. A derivative carrying the double substitution (KDD160RDV) had the unique phenotype of maintaining Fn binding while abolishing invasin binding, indicating that this region of the receptor may determine substrate specificity. These data indicate that the integrin beta1 chain possesses a ligand binding site shared by invasin and Fn that can be altered by mutation to allow greater preference for the normally lower affinity substrate Fn than for invasin. It was also established that the region analysed has the ability to bind divalent cations.     

Phosphotyrosine binding domains of Shc and insulin receptor substrate 1 recognize the NPXpY motif in a thermodynamically distinct manner

Phosphotyrosine binding (PTB) domains of the adaptor protein Shc and insulin receptor substrate (IRS-1) interact with a distinct set of activated and tyrosine-phosphorylated cytokine and growth factor receptors and play important roles in mediating mitogenic signal transduction. By using the technique of isothermal titration calorimetry, we have studied the thermodynamics of binding of the Shc and IRS-1 PTB domains to tyrosine-phosphorylated NPXY-containing peptides derived from known receptor binding sites. The results showed that relative contributions of enthalpy and entropy to the free energy of binding are dependent on specific phosphopeptides. Binding of the Shc PTB domain to tyrosine-phosphorylated peptides from TrkA, epidermal growth factor, ErbB3, and insulin receptors is achieved via an overall entropy-driven reaction. On the other hand, recognition of the phosphopeptides of insulin and interleukin-4 receptors by the IRS-1 PTB domain is predominantly an enthalpy-driven process. Mutagenesis and amino acid substitution experiments showed that in addition to the tyrosine-phosphorylated NPXY motif, the PTB domains of Shc and IRS-1 prefer a large hydrophobic residue at pY-5 and a small hydrophobic residue at pY-1, respectively (where pY is phosphotyrosine). These results agree with the calculated solvent accessibility of these two key peptide residues in the PTB domain/peptide structures and support the notion that the PTB domains of Shc and IRS-1 employ functionally distinct mechanisms to recognize tyrosine-phosphorylated receptors.     

Substrate recognition domains within extracellular signal-regulated kinase mediate binding and catalytic activation of mitogen-activated protein kinase phosphatase-3

Mitogen-activated protein (MAP) kinase phosphatase-3 (MKP-3) is a dual specificity phosphatase that inactivates extracellular signal-regulated kinase (ERK) MAP kinases. This reflects tight and specific binding between ERK and the MKP-3 amino terminus with consequent phosphatase activation and dephosphorylation of the bound MAP kinase. We have used a series of p38/ERK chimeric molecules to identify domains within ERK necessary for binding and catalytic activation of MKP-3. These studies demonstrate that ERK kinase subdomains V-XI are necessary and sufficient for binding and catalytic activation of MKP-3. These domains constitute the major COOH-terminal structural lobe of ERK. p38/ERK chimeras possessing these regions display increased sensitivity to inactivation by MKP-3. These data also reveal an overlap between ERK domains interacting with MKP-3 and those known to confer substrate specificity on the ERK MAP kinase. Consistent with this, we show that peptides representing docking sites within the target substrates Elk-1 and p90(rsk) inhibit ERK-dependent activation of MKP-3. In addition, abolition of ERK-dependent phosphatase activation following mutation of a putative kinase interaction motif (KIM) within the MKP-3 NH(2) terminus suggests that key sites of contact for the ERK COOH-terminal structural lobe include residues localized between the Cdc25 homology domains (CH2) found conserved between members of the DSP gene family.     

BiP and zein binding domains within the delta zein protein

Zeins are alcohol soluble seed storage proteins synthesized within the endosperm of maize and subsequently deposited into endoplasmic reticulum (ER) derived protein bodies. The genes encoding the beta and delta zeins were previously introduced into tobacco with the expectation of improving the nutritional quality of plants (Bagga et al. in Plant Physiol 107:13, 1997). Novel protein bodies are produced in the leaves of transgenic plants accumulating the beta or delta zein proteins. The mechanism of protein body formation within leaves is unknown. It is also unknown how zeins are retained in the ER since they do not contain known ER retention motifs. Retention may be due to an interaction of zeins with an ER chaperone such as binding luminal protein (BiP). We have demonstrated protein–protein interactions with the delta zeins, beta zeins, and BiP proteins using an E. coli two-hybrid system. In this study, four putative BiP binding motifs were identified within the delta zein protein using a BiP scoring program (Blond-Elguindi et al. in Cell 75:717, 1993). These putative binding motifs were mutated and their effects on protein interactions were analyzed in both a prokaryotic two-hybrid system and in plants. These mutations resulted in reduced BiP–zein protein interaction and also altered zein–zein interactions. Our results indicate that specific motifs are necessary for BiP–delta zein protein interactions and that there are specific motifs which are necessary for zein–zein interactions. Furthermore, our data demonstrates that zein proteins must be able to interact with BiP and zeins for their stability and ability to form protein bodies.     

Structure and ligand recognition of the PB1 domain: a novel protein module binding to the PC motif

PB1 domains are novel protein modules capable of binding to target proteins that contain PC motifs. We report here the NMR structure and ligand-binding site of the PB1 domain of the cell polarity establishment protein, Bem1p. In addition, we identify the topology of the PC motif-containing region of Cdc24p by NMR, another cell polarity establishment protein that interacts with Bem1p. The PC motif-containing region is a structural domain offering a scaffold to the PC motif. The chemical shift perturbation experiment and the mutagenesis study show that the PC motif is a major structural element that binds to the PB1 domain. A structural database search reveals close similarity between the Bem1p PB1 domain and the c-Raf1 Ras-binding domain. However, these domains are functionally distinct from each other.     

A 64 kd nuclear protein binds to RNA segments that include the AAUAAA polyadenylation motif

A 64 kd protein was shown to bind to RNAs that contain functional polyadenylation signals by a UV cross-linking procedure in which label was transferred from RNA substrate to protein in cell-free polyadenylation extracts. The 64 kd nuclear protein bound specifically to three different substrates (adenovirus type 5 L3, SV40 early, and SV40 late polyadenylation domains), as determined by competition experiments and partial protease analysis. Deleted derivatives of the SV40 late substrate that retained the sequence 5'-CUGCAAUAAACAAGUU-3' were able to bind the 64 kd polypeptide. This sequence contains the canonical AAUAAA element that has been shown to be indispensable for polyadenylation. A single nucleotide change, converting AAUAAA to AAGAAA, prevented binding of the 64 kd moiety. The 64 kd protein was shown to be distinct from poly(A) polymerase by biochemical fractionation.     

Integrin and neurocan binding to L1 involves distinct Ig domains.

The cell adhesion molecule L1, a 200-220-kDa type I membrane glycoprotein of the Ig superfamily, mediates many neuronal processes. Originally studied in the nervous system, L1 is expressed by hematopoietic and many epithelial cells, suggesting a more expanded role. L1 supports homophilic L1-L1 and integrin-mediated cell binding and can also bind with high affinity to the neural proteoglycan neurocan; however, the binding site is unknown. We have dissected the L1 molecule and investigated the cell binding ability of Ig domains 1 and 6. We report that RGD sites in domain 6 support alpha5beta1- or alphavbeta3-mediated integrin binding and that both RGD sites are essential. Cooperation of RGD sites with neighboring domains are necessary for alpha(5)beta(1). A T cell hybridoma and activated T cells could bind to L1 in the absence of RGDs. This binding was supported by Ig domain 1 and mediated by cell surface-exposed neurocan. Lymphoid and brain-derived neurocan were structurally similar. We also present evidence that a fusion protein of the Ig 1-like domain of L1 can bind to recombinant neurocan. Our results support the notion that L1 provides distinct cell binding sites that may serve in cell-cell or cell-matrix interactions.     

Protein phosphatase-1 activation and association with the retinoblastoma protein in colcemid-induced apoptosis

Protein phosphatase-1 (PP1) is cell cycle regulated and potentially related to apoptosis. We studied PP1 in HeLa cells exposed to colcemid, which leads first to mitotic block, then to cell death within 72 h. The soluble PP1 activity, which was low at 14 h (mitosis), was then reversibly activated (maximally around 48 h), with parallel changes in the protein levels of the alpha, gamma1 and delta PP1 isoforms. PP1 activation suggested its involvement in dephosphorylating proteins relevant to apoptosis. Among these, we examined the retinoblastoma protein (pRb). This was found hyperphosphorylated at 14 h. Hypophosphorylated pRb appeared at 24 h, increased at 48 h, and was the only form left at 72 h. PP1 was found to associate with immunoprecipitated pRb, as indicated by PP1 activity assays on the pRb-immunocomplexes. The pRb-associated PP1 activity was low at 14 h, maximal at 24 h, low again by 72 h and was due to PP1delta. The presence of active PP1 suggests its involvement in pRb dephosphorylation.     

Mutants of phosphorylase a altered in recognition by protein phosphatase-1

To develop our knowledge of specificity determinants for protein phosphatase-1, mutants of phosphorylase b have been converted to phosphorylase a and examined for their efficacy as substrates for protein phosphatase-1. Mutants focused on the N-terminal primary sequence surrounding the phosphoserine (R16A, R16E, and I13G) and at a site that interacts with the phosphoserine in phosphorylase a, (R69K and R69E). The success achieved studying protein kinase substrate specificity with peptide substrates has not extended to protein phosphatases. Protein phosphatases are believed to recognize higher order structure in substrates in addition to the primary sequence surrounding the phosphoserine or threonine. Peptide studies with protein phosphatase-1 have revealed a preference for basic residues N-terminal to the phosphoserine. Arginine 16 in phosphorylase a may be a positive determinant. In this work, protein phosphatase-1 preferred the positive charge on arginine 16. R16A exhibited a similar K(m) but reduced V(max), and R16E had an increased K(m) and a decreased V(max) when compared to phosphorylase. I13G had a similar K(m) but an increased V(max). The R69 mutants were also dephosphorylated preferentially over phosphorylase a. The K(m) for R69K was unchanged but had a higher V(max). R69E exhibited the most changes, with a 4-fold increase in K(m) and a 10-fold increase in V(max). These results suggest that proper presentation of the phosphoserine can greatly affect the rate of dephosphorylation.     

The role of the C-terminal domain of protein tyrosine phosphatase-1B in phosphatase activity and substrate binding

Protein tyrosine phosphatase 1B (PTP-1B) has been implicated in the regulation of the insulin receptor. Dephosphorylation of the insulin receptor results in decreased insulin signaling and thus decreased glucose uptake. PTP-1B-/- mice have increased insulin sensitivity and are resistant to weight gain when fed a high fat diet, validating PTP-1B as a potential target for the treatment of type 2 diabetes. Many groups throughout the world have been searching for selective inhibitors for PTP-1B, and most of them target inhibitors to PTP-1B-(1-298), the N-terminal catalytic domain of the enzyme. However, the C-terminal domain is quite large and could influence the activity of the enzyme. Using two constructs of PTP-1B and a phosphopeptide as substrate, steady state assays showed that the presence of the C-terminal domain decreased both the Km and the k(cat) 2-fold. Pre-steady state kinetic experiments showed that the presence of the C-terminal domain improved the affinity of the enzyme for a phosphopeptide 2-fold, primarily because the off-rate was slower. This suggests that the C-terminal domain of PTP-1B may contact the phosphopeptide in some manner, allowing it to remain at the active site longer. This could be useful when screening libraries of compounds for inhibitors of PTP-1B. A compound that is able to make contacts with the C-terminal domain of PTP-1B would not only have a modest improvement in affinity but may also provide for specificity over other phosphatases.     

Evidence that binding of cucumber necrosis virus to vector zoospores involves recognition of oligosaccharides

Despite the importance of vectors in natural dissemination of plant viruses, relatively little is known about the molecular features of viruses and vectors that permit their interaction in nature. Cucumber necrosis virus (CNV) is a small spherical virus whose transmission in nature is facilitated by zoospores of the fungus Olpidium bornovanus. Previous studies have shown that specific regions of the CNV capsid are involved in transmission and that transmission defects in several CNV transmission mutants are due to inefficient attachment of virions to the zoospore surface. In this study, we have undertaken to determine if zoospores contain specific receptors for CNV. We show that in vitro binding of CNV to zoospores is saturable and that vector zoospores bind CNV more efficiently than nonvector zoospores. Further studies show that treatment of zoospores with periodate and trypsin reduces CNV binding, suggesting the involvement of glycoproteins in zoospore attachment. In virus overlay assays, CNV binds to several proteins, whereas CNV transmission mutants either fail to bind or bind at significantly reduced levels. The possible involvement of specific sugars in attachment was investigated by incubating CNV with zoospores in the presence of various sugars. Two mannose derivatives (methyl alpha-D-mannopyranoside and D-mannosamine), as well as three mannose-containing oligosaccharides (mannotriose, alpha3,alpha6-mannopentaose, and yeast mannan) and L-(-)-fucose, all inhibited CNV binding at relatively low concentrations. Taken together, our studies suggest that binding of CNV to zoospores is mediated by specific mannose and/or fucose-containing oligosaccharides. This is the first time sugars have been implicated in transmission of a plant virus.     

Identification of a novel Mcl-1 protein binding motif

Recent characterization of Mcl-1 as the primary anti-apoptotic Bcl-2 family member expressed in solid tumors, coupled with its ability to enable therapeutic resistance, has provided the impetus for further study into how Mcl-1 is involved in apoptosis signaling. Here, we employ Sabutoclax, a potent and effective Mcl-1 antagonist, as a competing agent to screen a randomized 12-residue phage display library for peptides that bind strongly to the Bcl-2 homology 3 (BH3) binding groove of Mcl-1. Although the screen identified a number of α-helical peptides with canonical BH3 domain sequences, it also isolated a pair of unique peptide sequences. These sequences exhibit a reverse organization of conserved hydrophobic and acidic residues when compared with canonical BH3 sequences, and we therefore refer to them as reverse BH3 (rBH3) peptides. Furthermore, studies of the rBH3 peptides using NMR spectroscopy, fluorescence polarization displacement assays, and alanine scanning data all suggest that they bind to the BH3 binding groove of Mcl-1 selectively over Bcl-x(L). A search for proteins containing the rBH3 motif has identified a number of interesting Mcl-1 protein partners, some of which have previously been associated with apoptosis regulation involving Mcl-1. These findings provide insights into the development of more specific Mcl-1 antagonists and open the way to the identification of a previously unknown family of apoptosis-regulating and Mcl-1 interacting proteins.     

Specificity in substrate binding by protein folding catalysts: tyrosine and tryptophan residues are the recognition motifs for the binding of peptides to the pancreas-specific protein disulfide isomerase PDIp

Using a cross-linking approach, we recently demonstrated that radiolabeled peptides or misfolded proteins specifically interact in vitro with two luminal proteins in crude extracts from pancreas microsomes. The proteins were the folding catalysts protein disulfide isomerase (PDI) and PDIp, a glycosylated, PDI-related protein, expressed exclusively in the pancreas. In this study, we explore the specificity of these proteins in binding peptides and related ligands and show that tyrosine and tryptophan residues in peptides are the recognition motifs for their binding by PDIp. This peptide-binding specificity may reflect the selectivity of PDIp in binding regions of unfolded polypeptide during catalysis of protein folding.     

Identification of a novel phosphatidic acid binding domain in protein phosphatase-1

Phosphatidic acid (PA) has been recognized as a lipid second messenger, yet few cellular targets for PA have been identified. Previous work demonstrated PA as a potent and noncompetitive tight-binding inhibitor of the catalytic subunit (gamma isoform) of protein phosphatase-1 (PP1c gamma) in vitro. The high potency of inhibition, coupled with high specificity for PA over other phospholipids, suggested the presence of a high-affinity PA binding domain on PP1c gamma. In the current study, quantification of the binding interaction and identification of the binding domain were pursued. Surface plasmon resonance was employed to quantitate the interaction between PP1c gamma and immobilized mixed lipid vesicles of PA/phosphatidylcholine (PC) or PC alone. The data disclosed a high-affinity interaction with a KD measured in the low (1-40) nanomolar range, consistent with the range of Ki previously obtained from in vitro enzymatic assays. Next, identification of the segment of PP1 necessary for PA binding was determined using a deletion mutagenesis strategy. Binding assays revealed that PP1c gamma residues between 274 and 299 were required for the interaction with the lipid. When fusions of PP1c gamma fragments with green fluorescent protein (GFP) were generated, it was then determined that PP1c gamma residues 286-296 were sufficient to confer PA binding to GFP, a protein that does not interact with PA. The minimal PA binding domain of PP1c gamma lacked similarity to the previously described PA binding segments of Raf-1 kinase and cyclic-AMP phosphodiesterase 4A1. When these results were taken together with the known crystallographic structure of PP1, they identified a novel PA binding region on PP1c gamma that contains a unique loop-strand structural fold responsible for the interaction with PA.     

Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1.

The Hsp70 family of molecular chaperones has an essential role in the synthesis, folding and translocation of the nascent peptide chain. While the general features of these activities are well documented, less is understood about the regulation of these activities. The ATPase rate is stimulated by non-native proteins, furthermore, interaction with ATP leads to the release of protein substrate concurrent with a conformational change in Hsp70. One interpretation of these data is that the two domains of Hsp70 interact. In the process of mapping the carboxyl-terminal boundary of the substrate binding domain for human Hsp70, we identified a regulatory motif, EEVD, which is conserved at the extreme carboxyl terminus among nearly all cloned cytosolic eukaryotic Hsp70s. Deletion or mutation of EEVD affects the ATPase activity, the ability to interact with substrates, and interferes with the ability of the mutant Hsp70 to interact with HDJ-1 in the refolding of denatured firefly luciferase. Examination of the biophysical properties of the mutant Hsp70s reveals a change in the overall shape and conformation of the protein consistent with reduced interactions between the two domains. These data suggest that the EEVD motif is involved in the intramolecular regulation of Hsp70 function and intermolecular interactions with HDJ-1.