Improved affinity of engineered streptavidin for the Strep-tag II peptide is due to a fixed open conformation of the lid-like loop at the binding site

The Strep-tag II is a nine-amino acid peptide that was developed as an affinity tool for the purification of corresponding fusion proteins on streptavidin columns. The peptide recognizes the same pocket of streptavidin where the natural ligand is normally bound so that biotin or its chemical derivatives can be used for competitive elution. We report here the crystal structures of the streptavidin mutants '1' and '2,' which had been engineered for 10-fold higher affinity towards the Strep-tag II. Both streptavidin mutants carry mutations at positions 44, 45, and 47, that is, in a flexible loop region close to the binding site. The crystal structures of the two apo-proteins and their complexes with the Strep-tag II peptide were refined at resolutions below 2 A. Both in the presence and absence of the peptide, the lid-like loop next to the ligand pocket--comprising residues 45 through 52--adopts an 'open' conformation in all four subunits within the asymmetric unit. The same loop was previously described to be disordered in the wild-type apo-streptavidin and to close over the pocket upon complexation of the natural ligand biotin. Our findings suggest that stabilization of the 'open' loop conformation in the absence of a ligand abolishes the need for conformational rearrangement prior to the docking of the voluminous peptide. Because no direct contacts between the flexible part of the loop and the peptide ligand were detected, it seems likely that the higher affinity of the two streptavidin mutants for the Strep-tag II is caused by a predominantly entropic mechanism.     

Use of a biomimetic peptide in the design of a competitive binding assay for biotin and biotin analogues

A competitive binding assay for biotin, biocytin, and desthiobiotin utilizing a genetically engineered enzyme-ligand conjugate is described herein. This assay is unique in that the enzyme-ligand conjugate consists of the streptavidin binding peptide Strep-tag II, which mimics the binding of biotin to streptavidin, rather than biotin itself. This allows for the construction of a well-defined, oligosubstituted enzyme-ligand conjugate for which the site of attachment of the ligand on the enzyme is known precisely. The assay has detection limits of 5 x 10(-8) M for biotin, 1 x 10(-7) M for biocytin, and 2 x 10(-6) M for desthiobiotin, and it serves as a model system in that it demonstrates the feasibility of using enzyme-ligand conjugates in which a peptide mimic of the analyte ligand is genetically fused to the enzyme. This avoids the problems associated with covalent attachment of the ligand to the enzyme, such as multiple substitution of the ligand and variability of the site of attachment. To our knowledge, this is the first example of using an enzyme-peptide mimic conjugate to detect a nonpeptide analyte.     

Easily reversible desthiobiotin binding to streptavidin,avidin, and other biotin-binding proteins: uses for protein labeling,detection, and isolation

The high-affinity binding of biotin to avidin, streptavidin, and related proteins has been exploited for decades. However, a disadvantage of the biotin/biotin-binding protein interaction is that it is essentially irreversible under physiological conditions. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by biotin. We synthesized an amine-reactive desthiobiotin derivative for labeling proteins and a desthiobiotin-agarose affinity matrix. Conjugates labeled with desthiobiotin are equivalent to their biotinylated counterparts in cell-staining and antigen-labeling applications. They also bind to streptavidin and other biotin-binding protein-based affinity columns and are recognized by anti-biotin antibodies. Fluorescent streptavidin conjugates saturated with desthiobiotin, but not biotin, bind to a cell-bound biotinylated target without further processing. Streptavidin-based ligands can be gently stripped from desthiobiotin-labeled targets with buffered biotin solutions. Thus, repeated probing with fluorescent streptavidin conjugates followed by enzyme-based detection is possible. In all applications, the desthiobiotin/biotin-binding protein complex is easily dissociated under physiological conditions by either biotin or desthiobiotin. Thus, our desthiobiotin-based reagents and techniques provide some distinct advantages over traditional 2-iminobiotin, monomeric avidin, or other affinity-based techniques.     

X-ray crystallographic studies of streptavidin mutants binding to biotin

On the basis of high resolution crystallographic studies of streptavidin and its biotin complex, three principal binding motifs have been identified that contribute to the tight binding. A flexible binding loop can undergo a conformational change from an open to a closed form when biotin is bound. Additional studies described here of unbound wild-type streptavidin have provided structural views of the open conformation. Several tryptophan residues packing around the bound biotin constitute the second binding motif, one dominated by hydrophobic interactions. Mutation of these residues to alanine or phenylalanine have variable effects on the thermodynamics and kinetics of binding, but they generate only small changes in the molecular structure. Hydrogen bonding interactions also contribute significantly to the binding energetics of biotin, and the D128A mutation which breaks a hydrogen bond between the protein and a ureido NH group results in a significant structural alteration that could mimic an intermediate on the dissociation pathway. In this review, we summarize the structural aspects of biotin recognition that have been gained from crystallographic analyses of wild-type and site-directed streptavidin mutants.     

The Solution Structure,Binding Properties,and Dynamics of the Bacterial Siderophore-binding Protein FepB

The periplasmic binding protein (PBP) FepB plays a key role in transporting the catecholate siderophore ferric enterobactin from the outer to the inner membrane in Gram-negative bacteria. The solution structures of the 34-kDa apo- and holo-FepB from Escherichia coli, solved by NMR, represent the first solution structures determined for the type III class of PBPs. Unlike type I and II PBPs, which undergo large “Venus flytrap” conformational changes upon ligand binding, both forms of FepB maintain similar overall folds; however, binding of the ligand is accompanied by significant loop movements. Reverse methyl cross-saturation experiments corroborated chemical shift perturbation results and uniquely defined the binding pocket for gallium enterobactin (GaEnt). NMR relaxation experiments indicated that a flexible loop (residues 225–250) adopted a more rigid and extended conformation upon ligand binding, which positioned residues for optimal interactions with the ligand and the cytoplasmic membrane ABC transporter (FepCD), respectively. In conclusion, this work highlights the pivotal role that structural dynamics plays in ligand binding and transporter interactions in type III PBPs.     

Structural studies of the streptavidin binding loop.

The streptavidin-biotin complex provides the basis for many important biotechnological applications and is an interesting model system for studying high-affinity protein-ligand interactions. We report here crystallographic studies elucidating the conformation of the flexible binding loop of streptavidin (residues 45 to 52) in the unbound and bound forms. The crystal structures of unbound streptavidin have been determined in two monoclinic crystal forms. The binding loop generally adopts an open conformation in the unbound species. In one subunit of one crystal form, the flexible loop adopts the closed conformation and an analysis of packing interactions suggests that protein-protein contacts stabilize the closed loop conformation. In the other crystal form all loops adopt an open conformation. Co-crystallization of streptavidin and biotin resulted in two additional, different crystal forms, with ligand bound in all four binding sites of the first crystal form and biotin bound in only two subunits in a second. The major change associated with binding of biotin is the closure of the surface loop incorporating residues 45 to 52. Residues 49 to 52 display a 3(10) helical conformation in unbound subunits of our structures as opposed to the disordered loops observed in other structure determinations of streptavidin. In addition, the open conformation is stabilized by a beta-sheet hydrogen bond between residues 45 and 52, which cannot occur in the closed conformation. The 3(10) helix is observed in nearly all unbound subunits of both the co-crystallized and ligand-free structures. An analysis of the temperature factors of the binding loop regions suggests that the mobility of the closed loops in the complexed structures is lower than in the open loops of the ligand-free structures. The two biotin bound subunits in the tetramer found in the MONO-b1 crystal form are those that contribute Trp 120 across their respective binding pockets, suggesting a structural link between these binding sites in the tetramer. However, there are no obvious signatures of binding site communication observed upon ligand binding, such as quaternary structure changes or shifts in the region of Trp 120. These studies demonstrate that while crystallographic packing interactions can stabilize both the open and closed forms of the flexible loop, in their absence the loop is open in the unbound state and closed in the presence of biotin. If present in solution, the helical structure in the open loop conformation could moderate the entropic penalty associated with biotin binding by contributing an order-to-disorder component to the loop closure.     

Development of a tetrameric streptavidin mutein with reversible biotin binding capability: engineering a mobile loop as an exit door for biotin

A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop(3-4) functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop(3-4) keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop(7-8). This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop(7-8) is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (k(off) of 4.28×10(-4) s(-1) and K(d) of 1.9×10(-8) M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible.     

The Role of Changing Loop Conformations in Streptavidin Versions Engineered for High-affinity Binding of the Strep-tag II Peptide

The affinity system based on the artificial peptide ligand Strep-tag® II and engineered tetrameric streptavidin, known as Strep-Tactin®, offers attractive applications for the study of recombinant proteins, from detection and purification to functional immobilization. To further improve binding of the Strep-tag II to streptavidin we have subjected two protruding loops that shape its ligand pocket for the peptide – instead of D-biotin recognized by the natural protein – to iterative random mutagenesis. Sequence analyses of hits from functional screening assays revealed several unexpected structural motifs, such as a disulfide bridge at the base of one loop, replacement of the crucial residue Trp120 by Gly and a two-residue deletion in the second loop. The mutant m1-9 (dubbed Strep-Tactin XT) showed strongly enhanced affinity towards the Strep-tag II, which was further boosted in case of the bivalent Twin-Strep-tag®. Four representative streptavidin mutants were crystallized in complex with the Strep-tag II peptide and their X-ray structures were solved at high resolutions. In addition, the crystal structure of the complex between Strep-Tactin XT and the Twin-Strep-tag was elucidated, indicating a bivalent mode of binding and explaining the experimentally observed avidity effect. Our study illustrates the structural plasticity of streptavidin as a scaffold for ligand binding and reveals interaction modes that would have been difficult to predict. As result, Strep-Tactin XT offers a convenient reagent for the kinetically stable immobilization of recombinant proteins fused with the Twin-Strep-tag. The possibility of reversibly dissociating such complexes simply with D-biotin as a competing ligand enables functional studies in protein science as well as cell biology.     

Observation of signal transduction in three-dimensional domain swapping

p13suc1 (suc1) has two native states, a monomer and a domain-swapped dimer. The structure of each subunit in the dimer is identical to that of the monomer, except for the hinge loop that connects the exchanging domains. Here we find that single point mutations at sites throughout the protein and ligand binding both shift the position of the equilibrium between monomer and dimer. The hinge loop was shown previously to act as a loaded molecular spring that releases tension present in the monomer by adopting an alternative conformation in the dimer. The results here indicate that the release of strain propagates throughout the entire protein and alters the energetics of regions remote from the hinge. Our data illustrate how the signal conferred by the conformational change of a protein loop, elicited by domain swapping, ligand binding or mutation, can be sensed by a distant active site. This work highlights the potential role of strained loops in proteins: the energy they store can be used for both signal transduction and allostery, and they could steer the evolution of protein function. Finally, a structural mechanism for the role of suc1 as an adapter molecule is proposed.     

The highly dynamic oligomeric structure of bradavidin II is unique among avidin proteins

Bradavidin II is a biotin‐binding protein from Bradyrhizobium japonicum that resembles chicken avidin and bacterial streptavidin. A biophysical characterization was carried out using dynamic light scattering, native mass spectrometry, differential scanning calorimetry, and isothermal titration calorimetry combined with structural characterization using X‐ray crystallography. These observations revealed that bradavidin II differs from canonical homotetrameric avidin protein family members in its quaternary structure. In contrast with the other avidins, bradavidin II appears to have a dynamic (transient) oligomeric state in solution. It is monomeric at low protein concentrations but forms higher oligomeric assemblies at higher concentrations. The crystal structure of bradavidin II revealed an important role for Phe42 in shielding the bound ligand from surrounding water molecules, thus functionally replacing the L7,8 loop essential for tight ligand binding in avidin and streptavidin. This bradavidin II characterization opens new avenues for oligomerization‐independent biotin‐binding protein development.     

Structure‐based engineering of streptavidin monomer with a reduced biotin dissociation rate

We recently reported the engineering of monomeric streptavidin, mSA, corresponding to one subunit of wild type (wt) streptavidin tetramer. The monomer was designed by homology modeling, in which the streptavidin and rhizavidin sequences were combined to engineer a high affinity binding pocket containing residues from a single subunit only. Although mSA is stable and binds biotin with nanomolar affinity, its fast off rate (k_off) creates practical challenges during applications. We obtained a 1.9 Å crystal structure of mSA bound to biotin to understand their interaction in detail, and used the structure to introduce targeted mutations to improve its binding kinetics. To this end, we compared mSA to shwanavidin, which contains a hydrophobic lid containing F43 in the binding pocket and binds biotin tightly. However, the T48F mutation in mSA, which introduces a comparable hydrophobic lid, only resulted in a modest 20–40% improvement in the measured k_off. On the other hand, introducing the S25H mutation near the bicyclic ring of bound biotin increased the dissociation half life (t_½) from 11 to 83 min at 20°C. Molecular dynamics (MD) simulations suggest that H25 stabilizes the binding loop L3,4 by interacting with A47, and protects key intermolecular hydrogen bonds by limiting solvent entry into the binding pocket. Concurrent T48F or T48W mutation clashes with H25 and partially abrogates the beneficial effects of H25. Taken together, this study suggests that stabilization of the binding loop and solvation of the binding pocket are important determinants of the dissociation kinetics in mSA. Proteins 2013. © 2013 Wiley Periodicals, Inc.     

How different are structurally flexible and rigid binding sites? Sequence and structural features discriminating proteins that do and do not undergo conformational change upon ligand binding

Proteins are dynamic molecules and often undergo conformational change upon ligand binding. It is widely accepted that flexible loop regions have a critical functional role in enzymes. Lack of consideration of binding site flexibility has led to failures in predicting protein functions and in successfully docking ligands with protein receptors. Here we address the question: which sequence and structural features distinguish the structurally flexible and rigid binding sites? We analyze high-resolution crystal structures of ligand bound (holo) and free (apo) forms of 41 proteins where no conformational change takes place upon ligand binding, 35 examples with moderate conformational change, and 22 cases where a large conformational change has been observed. We find that the number of residue-residue contacts observed per-residue (contact density) does not distinguish flexible and rigid binding sites, suggesting a role for specific interactions and amino acids in modulating the conformational changes. Examination of hydrogen bonding and hydrophobic interactions reveals that cases that do not undergo conformational change have high polar interactions constituting the binding pockets. Intriguingly, the large, aromatic amino acid tryptophan has a high propensity to occur at the binding sites of examples where a large conformational change has been noted. Further, in large conformational change examples, hydrophobic-hydrophobic, aromatic-aromatic, and hydrophobic-polar residue pair interactions are dominant. Further analysis of the Ramachandran dihedral angles (phi, psi) reveals that the residues adopting disallowed conformations are found in both rigid and flexible cases. More importantly, the binding site residues adopting disallowed conformations clustered narrowly into two specific regions of the L-Ala Ramachandran map. Examination of the dihedral angles changes upon ligand binding shows that the magnitude of phi, psi changes are in general minimal, although some large changes particularly between right-handed alpha-helical and extended conformations are seen. Our work further provides an account of conformational changes in the dihedral angles space. The findings reported here are expected to assist in providing a framework for predicting protein-ligand complexes and for template-based prediction of protein function.     

High resolution structure of streptavidin in complex with a novel high affinity peptide tag mimicking the biotin binding motif

A novel peptide was designed which possesses nanomolar affinity of less than 20 nM for streptavidin. Therefore it was termed Nano-tag and has been used as an affinity tag for recombinant proteins. The minimized version of the wild type Nano-tag is a seven-amino acid peptide with the sequence fMDVEAWL. The three-dimensional structure of wild type streptavidin in complex with the minimized Nano-tag was analyzed at atomic resolution of 1.15 A and the details of the binding motif were investigated. The peptide recognizes the same pocket of streptavidin where the natural ligand biotin is bound, but the peptide requires significantly more space than biotin. Therefore the binding loop adopts an "open" conformation in order to release additional space for the peptide. The conformation of the bound Nano-tag corresponds to a 3(10) helix. However, the analysis of the intermolecular interactions of the Nano-tag with residues of the binding pocket of streptavidin reveals astonishing similarities to the biotin binding motif. In principle the three-dimensional conformation of the Nano-tag mimics the binding mode of biotin. Our results explain why the use of the Nano-tag in fusion with recombinant proteins is restricted to their N-terminus and we describe the special significance of the fMet residue for the high affinity binding mode.     

Photocrosslinking/label transfer: A key step in mapping short alpha-neurotoxin binding site on nicotinic acetylcholine receptor

We developed a novel radioactive short bifunctional photoprobe, which could be coupled through a cleavable bond to an engineered cysteinyl residue on an analogue of a nicotinic acetylcholine receptor-specific alpha-neurotoxin. This cysteine was put on the tip of loop II in place of Arg33, a major residue for the interaction with the receptor. To facilitate the purification of the nAChR labeled subunits, we tagged the ligand with a desthiobiotin moiety. After irradiation of the photosensitive toxin-nAChR complex, gel electrophoresis showed that most of the radioactivity was attached to the alpha subunit (59%), followed by the gamma subunit (28%), with the delta subunit (13%) being less labeled. On a preparative scale, the labeled subunits were purified on streptavidin beads before separation on SDS-PAGE. "In-gel" CNBr cleavage of the labeled alpha subunit followed by Edman degradation of the purified peptides showed that alphaTyr190 and alphaTyr198 were the most labeled residues, with a less important labeling on alphaCys192. We believe that the novel photoactivatable probe will be of great use to identify key residues of ligands interacting with macromolecules.     

Molecular dynamics simulation of PNPLA3 I148M polymorphism reveals reduced substrate access to the catalytic cavity

A missense mutation I148M in PNPLA3 (patatin‐like phospholipase domain‐containing 3 protein) is significantly correlated with nonalcoholic fatty liver disease (NAFLD). To glean insights into mutation's effect on enzymatic activity, we performed molecular dynamics simulation and flexible docking studies. Our data show that the size of the substrate‐access entry site is significantly reduced in mutants, which limits the access of palmitic acid to the catalytic dyad. Besides, the binding free energy calculations suggest low affinity for substrate to mutant enzyme. The substrate‐bound system simulations reveal that the spatial arrangement of palmitic acid is distinct in wild‐type from that in mutant. The substrate recognition specificity is lost due to the loop where the I148M mutation was located. Our results provide strong evidence for the mechanism by which I148M affects the enzyme activity and suggest that mediating the dynamics may offer a potential avenue for NAFLD. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Contributions to the binding free energy of ligands to avidin and streptavidin

The free energy of binding of a ligand to a macromolecule is here formally decomposed into the (effective) energy of interaction, reorganization energy of the ligand and the macromolecule, conformational entropy change of the ligand and the macromolecule, and translational and rotational entropy loss of the ligand. Molecular dynamics simulations with implicit solvation are used to evaluate these contributions in the binding of biotin, biotin analogs, and two peptides to avidin and streptavidin. We find that the largest contribution opposing binding is the protein reorganization energy, which is calculated to be from 10 to 30 kcal/mol for the ligands considered here. The ligand reorganization energy is also significant for flexible ligands. The translational/rotational entropy is 4.5-6 kcal/mol at 1 M standard state and room temperature. The calculated binding free energies are in the correct range, but the large statistical uncertainty in the protein reorganization energy precludes precise predictions. For some complexes, the simulations show multiple binding modes, different from the one observed in the crystal structure. This finding is probably due to deficiencies in the force field but may also reflect considerable ligand flexibility.     

Conformational and dynamics changes induced by bile acids binding to chicken liver bile acid binding protein

The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family.     

Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop_3–4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (K_d) from 4×10⁻¹⁴ M to 4.45×10⁻¹⁰ M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible.     

Negative factor from SIV binds to the catalytic subunit of the V-ATPase to internalize CD4 and to increase viral infectivity

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIV(mac239) for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity.     

Mutational activation of ErbB2 reveals a new protein kinase autoinhibition mechanism

Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the alphaC helix and beta4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to approximately 10- and approximately 7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the alphaC-beta4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the alphaC-beta4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the alphaC-beta4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of alphaC-beta4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2.