Allosteric activation of ADAMTS13 by von Willebrand factor

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) within endovascular platelet aggregates, and ADAMTS13 deficiency causes fatal microvascular thrombosis. The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains of ADAMTS13 recognize a cryptic site in VWF that is exposed by tensile force. Another seven T and two complement C1r/C1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains of uncertain function are C-terminal to the MDTCS domains. We find that the distal T8-CUB2 domains markedly inhibit substrate cleavage, and binding of VWF or monoclonal antibodies to distal ADAMTS13 domains relieves this autoinhibition. Small angle X-ray scattering data indicate that distal T-CUB domains interact with proximal MDTCS domains. Thus, ADAMTS13 is regulated by substrate-induced allosteric activation, which may optimize VWF cleavage under fluid shear stress in vivo. Distal domains of other ADAMTS proteases may have similar allosteric properties.After vascular injury, platelets adhere to von Willebrand factor (VWF) multimers bound to endothelial cell surfaces and connective tissue. The force of flowing blood on a growing platelet-rich thrombus stretches the central A2 domain of VWF and exposes a Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ cleavage site for ADAMTS13 (Fig. 1A) (1–5), a metalloprotease that severs VWF and releases adherent platelets. Deficiency of ADAMTS13 disrupts this feedback regulatory mechanism and causes thrombotic thrombocytopenic purpura (TTP), which is characterized by life-threatening microvascular thrombosis (3, 6, 7).Open in a separate windowFig. 1.Activation of ADAMTS13 by autoantibodies from a patient with TTP or by low pH. (A) Structure of ADAMTS13. (B) Fluorogenic substrates terminate at VWF residues indicated by arrows. Each substrate has Lys¹⁶¹⁷ replaced with Arg, N-terminal Gly modified with IRDye QC-1 (QC1), and Asn¹⁶¹⁰ replaced by Cys and modified with DyLight 633 (DyL) (22). The arrowhead indicates the cleaved Tyr-Met bond. Secondary structure elements of the VWF A2 domain (11) are indicated below and segments that interact with specific ADAMTS13 domains (13) are indicated above the sequence. (C) BCW49 plasma activated ADAMTS13 with a titer of 9.6 U at pH 7.4 (orange squares), but not at pH 6.0 (orange circle). BCW49 plasma did not activate MDTCS at pH 6 (blue circle) or pH 7.4 (blue circle). (D) Rates of VWF71 cleavage were determined as a function of pH for ADAMTS13 (orange circles) and MDTCS (blue circles). Error bars indicate 95% confidence intervals and if not shown are smaller than the symbols.The recognition and cleavage of VWF is a formidable challenge. VWF and ADAMTS13 occur at ∼10 µg/mL and ∼1 µg/mL, respectively, compared with total plasma protein of ∼80,000 µg/mL. ADAMTS13 is constitutively active and has no known inhibitors in vivo. Nevertheless, VWF is the only identified ADAMTS13 substrate, and VWF is resistant to cleavage until subjected to fluid shear stress (8), adsorbed on a surface (9), or treated with denaturants (8, 10). This specificity depends on structural features of both ADAMTS13 and VWF that have not been characterized fully.The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains domains of ADAMTS13 bind to cryptic sites that are uncovered by unfolding VWF domain A2 (11-15) (Fig. 1B), and these interactions are required for efficient cleavage of VWF or peptide substrates. More distal ADAMTS13 domains bind to sites in or near VWF domain D4 that are always available (16–18). Deletion of distal ADAMTS13 domains impairs the cleavage of VWF multimers in vitro (16, 19) and increases VWF-dependent microvascular thrombosis in vivo (20) but accelerates the cleavage of peptide substrates (12, 13). In addition, ADAMTS13 cleaves guanidine hydrochloride-treated VWF multimers with an apparent K_m of ∼15 nM (21), which is 100-fold lower than the K_m of ∼1.6–1.7 µM for peptide substrates that are based on the sequence of VWF domain A2 (12, 14). These striking differences suggest that distal T or complement c1r/c1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains regulate ADAMTS13 activity. We have now shown that these distal domains inhibit ADAMTS13, and binding to VWF relieves this autoinhibition.     

The loops of the N-SH2 binding cleft do not serve as allosteric switch in SHP2 activation

The Src-homology-2 domain–containing phosphatase SHP2 is a critical regulator of signal transduction, being implicated in cell growth and differentiation. Activating mutations cause developmental disorders and act as oncogenic drivers in hematologic cancers. SHP2 is activated by phosphopeptide binding to the N-SH2 domain, triggering the release of N-SH2 from the catalytic PTP domain. Based on early crystallographic data, it has been widely accepted that opening of the binding cleft of N-SH2 serves as the key “allosteric switch” driving SHP2 activation. To test the putative coupling between binding cleft opening and SHP2 activation as assumed by the allosteric switch model, we critically reviewed structural data of SHP2, and we used extensive molecular dynamics (MD) simulation and free energy calculations of isolated N-SH2 in solution, SHP2 in solution, and SHP2 in a crystal environment. Our results demonstrate that the binding cleft in N-SH2 is constitutively flexible and open in solution and that a closed cleft found in certain structures is a consequence of crystal contacts. The degree of opening of the binding cleft has only a negligible effect on the free energy of SHP2 activation. Instead, SHP2 activation is greatly favored by the opening of the central β-sheet of N-SH2. We conclude that opening of the N-SH2 binding cleft is not the key allosteric switch triggering SHP2 activation.

Src-homology-2–containing protein tyrosine phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a classical nonreceptor protein tyrosine phosphatase (PTP). It has emerged as a key downstream regulator of several receptor tyrosine kinases (RTKs) and cytokine receptors, functioning as a positive or negative modulator in multiple signaling pathways (1–4). Germline mutations in the human PTPN11 gene have been associated with Noonan syndrome and with Noonan syndrome with multiple lentigines (formerly known as LEOPARD syndrome), two multisystem developmental diseases (5–16). Somatic PTPN11 mutations were also linked with several types of human malignancies (17–25), such as myeloid leukemia (7, 26–30).The structure of SHP2 includes two tandemly arranged Src homology 2 domains (SH2), called N-SH2 and C-SH2, followed by the catalytic PTP domain, and a C-terminal tail with a poorly characterized function (Fig. 1) (31). The SH2 domains are structurally conserved recognition elements (32) that allow SHP2 to bind signaling peptides containing a phosphorylated tyrosine (pY) (33). The N-SH2 domain consists of a central antiparallel β-sheet, composed of three β-strands, denoted βB, βC, and βD, flanked by two α-helices, denoted αA and αB (Fig. 1A). The peptide binds in an extended conformation to the cleft that is perpendicular to the plane of the β-sheet (34). N-SH2 contains a conserved affinity pocket covered by the BC loop (also called phosphate-binding loop or pY loop), whose interaction with pY increases the binding of the peptide by 1,000-fold relative to unphosphorylated counterparts (35). Residues downstream of the pY bind to a more variable, less conserved site, which confers binding specificity and which is flanked by the EF and BG loops (36).Open in a separate windowFig. 1.(A) Cartoon representation of the N-SH2 domain in complex with the IRS-1 pY895 peptide (34). The peptide, shown in cyan, comprises the phosphotyrosine and residues from position +1 to +5 relative to the phosphotyrosine (see labels). Functionally important loops are highlighted in color: BC “pY” loop (green), DE “blocking loop” (light blue), EF loop (magenta), and BG loop (deep pink). The phosphotyrosine binds the site delimited by the pY loop and the central β-sheet (βB, βC, βD strands). EF and BG loops delimit the binding cleft (+5 site), where the peptide residue in position +5 is settled. (B) Crystal structure of autoinhibited SHP2 (37). The N-SH2 domain (cyan cartoon) blocks the catalytic site (red) of the PTP domain (pink) with the blocking loop (blue). The N-SH2 domain is connected to PTP in tandem with the homologous C-SH2 domain (orange). Closure of the N-SH2 binding cleft (green region), delimited by the EF and BG loops (magenta and deep pink), precludes high-affinity phosphopeptide binding. According to the “allosteric switch” model, the change in shape of the N-SH2 domain, which accompanies binding of phosphopeptide (yellow), perturbs surface complementarity for the PTP active site, thus promoting N-SH2 dissociation from the PTP domain. (C) Crystal structure of SHP2^E76K (38). The open conformation reveals a 120° rotation of the C-SH2 domain, relocation of the N-SH2 domain to a PTP surface opposite the active site, and a solvent-exposed catalytic pocket.In 1998, the first crystallographic structure of SHP2 at 2 Å resolution (Protein Data Bank [PDB] ID 2SHP) revealed that the N-SH2 domain tightly interacts with the PTP domain (Fig. 1B), so that the DE loop of N-SH2, thereafter indicated as “blocking loop,” occludes the active site of PTP, forcing SHP2 into an autoinhibited “closed” state (37). SHP2 structures of the active “open” state, obtained for the basally active, leukemia-associated E76K mutant, showed an alternative relative arrangement of N-SH2 and PTP that exposed the active site of the PTP domain to the solvent (Fig. 1C) (38). Unexpectedly, in both the autoinhibited and the active state of SHP2, the N-SH2 ligand binding site is exposed to solvent and does not directly interact with PTP or other domains (37–39). Hence, the need for an allosteric mechanism was proposed, according to which the binding of a phosphopeptide triggers a series of structural rearrangements in the N-SH2 domain to drive its release from PTP and the consequent activation of SHP2 (37, 39).The comparison of the autoinhibited structure of SHP2 (PDB ID 2SHP) (37) with the existing structures of the isolated N-SH2 domain, either in the absence of (PDB ID 1AYD) (34) or in complex with a phosphopeptide (PDB ID 1AYA, 1AYB, 4QSY) (34), showed that the EF and BG loops of the N-SH2 domain may undergo large conformational changes (Fig. 1B). In the autoinhibited structure of SHP2, the N-SH2 domain shares surface complementarity with the PTP catalytic site, but, as a result of the displacement of the EF loop toward the BG loop, it also contains a closed binding cleft that renders the N-SH2 domain unable to accommodate the C-terminal part of the phosphopeptide, in contrast to the isolated N-SH2 domain that exhibits an open binding cleft. Therefore, peptide binding seems only compatible with the conformation of isolated N-SH2, but not with the conformation of N-SH2 in autoinhibited SHP2 (37, 39).Because 1) the closure of the binding cleft in N-SH2 has been ascribed to its interaction with PTP and 2) the conformation adopted by the EF loop correlates with the activation of SHP2 in available structural data, the EF loop has been suggested as the key allosteric switch that drives the release of N-SH2 from PTP (37, 39). In light of that, conformation selection (9, 21, 40) and induced fit (41) models were put forward for the molecular events leading to functional activation of SHP2; however, both models consider the conformational change involving the EF loop as the key mechanism that drives SHP2 opening (9, 21, 40, 41). In conclusion, it has been widely accepted that the N-SH2 binding cleft, delimited by the EF and the BG loop, plays the role of an allosteric switch for the activation of SHP2 (39, 42). Accordingly, the binding of a ligand at the binding cleft of the N-SH2 domain would induce a transmitted conformational change that prevents PTP domain binding on the other side of N-SH2, and vice versa (37, 39).However, the allosteric switch model does not explain how the signal, coming from the displacement of the EF loop, is propagated to the rest of the protein (39). In addition, considering that the EF loop might be flexible, the comparison of crystallographic structures does not provide the energetic penalties involved in the motion of the EF loop and, consequently, the degree of destabilization of the N-SH2/PTP complex upon the binding cleft opening (39). Hence, the role of the EF loop as the key allosteric switch has not been rationalized in energetic terms.Recently, an allosteric interaction in N-SH2 has been proposed as an alternative mechanism of SHP2 activation (43). Molecular dynamics (MD) simulations have shown that the N-SH2 domain may adopt two distinct conformations, denoted as α- and β-states, which differ primarily in the conformation of the central β-sheet. In the α-state, the central β-sheet is open, adopting a Y-shaped structure; in the β-state, the central β-sheet is closed, adopting a parallel structure. The MD simulations suggested that the β-state of N-SH2 stabilizes the N-SH2/PTP contacts and, hence, the autoinhibited SHP2 conformation. In contrast, the α-state drives the N-SH2 dissociation and SHP2 activation. Notably, the α–β model of activation rationalized modified basal activity and responsiveness to ligand stimulation of certain mutations at codon 42 (15). However, the α–β model seems to contrast with the previously suggested allosteric switch model.To resolve this discrepancy, we revisited the allosteric switch model. We critically reviewed available crystallographic data of SHP2 in the autoinhibited state. In addition, we used MD simulations, free energy calculations, and enhanced sampling techniques to reveal the conformational dynamics of the binding cleft delimited by the BG and the EF loop in the isolated N-SH2 domain in water, in SHP2 in water, and in SHP2 in a crystal environment. Our results suggest that the binding cleft of N-SH2 is constitutively flexible and the effect of its degree of opening on the activation of SHP2 is negligible. In addition, free energy calculations revealed that, in the crystal environment, the closure of the binding cleft is not due to the allosteric interaction with the PTP domain, but instead a result of the crystal contacts affecting the binding cleft conformation.     

Interplay of PDZ and protease domain of DegP ensures efficient elimination of misfolded proteins

Aberrant proteins represent an extreme hazard to cells. Therefore, molecular chaperones and proteases have to carry out protein quality control in each cellular compartment. In contrast to the ATP-dependent cytosolic proteases and chaperones, the molecular mechanisms of extracytosolic factors are largely unknown. To address this question, we studied the protease function of DegP, the central housekeeping protein in the bacterial envelope. Our data reveal that DegP processively degrades misfolded proteins into peptides of defined size by employing a molecular ruler comprised of the PDZ1 domain and the proteolytic site. Furthermore, peptide binding to the PDZ domain transforms the resting protease into its active state. This allosteric activation mechanism ensures the regulated and rapid elimination of misfolded proteins upon folding stress. In comparison to the cytosolic proteases, the regulatory features of DegP are established by entirely different mechanisms reflecting the convergent evolution of an extracytosolic housekeeping protease.     

Feedback regulation of plastidic acetyl-CoA carboxylase by 18:1-acyl carrier protein in Brassica napus

Plant seed oil represents a major renewable source of reduced carbon, but little is known about the biochemical regulation of its synthesis. The goal of this research was to identify potential feedback regulation of fatty acid biosynthesis in Brassica napus embryo-derived cell cultures and to characterize both the feedback signals and enzymatic targets of the inhibition. Fatty acids delivered via Tween esters rapidly reduced the rate of fatty acid synthesis in a dose-dependent and reversible manner, demonstrating the existence of feedback inhibition in an oil-accumulating tissue. Tween feeding did not affect fatty acid elongation in the cytosol or the incorporation of radiolabeled malonate into nascent fatty acids, which together pinpoint plastidic acetyl-CoA carboxylase (ACCase) as the enzymatic target of feedback inhibition. To identify the signal responsible for feedback, a variety of Tween esters were tested for their effects on the rate of fatty acid synthesis. Maximum inhibition was achieved upon feeding oleic acid (18:1) Tween esters that resulted in the intracellular accumulation of 18:1 free fatty acid, 18:1-CoA, and 18:1-acyl-carrier protein (ACP). Direct, saturable inhibition of ACCase enzyme activity was observed in culture extracts and in extracts of developing canola seeds supplemented with 18:1-ACP at physiological concentrations. A mechanism for feedback inhibition is proposed in which reduced demand for de novo fatty acids results in the accumulation of 18:1-ACP, which directly inhibits plastidic ACCase, leading to reduced fatty acid synthesis. Defining this mechanism presents an opportunity for mitigating feedback inhibition of fatty acid synthesis in crop plants to increase oil yield.     

Allosteric gating mechanism underlies the flexible gating of KCNQ1 potassium channels

KCNQ1 (Kv7.1) is a unique member of the superfamily of voltage-gated K(+) channels in that it displays a remarkable range of gating behaviors tuned by coassembly with different β subunits of the KCNE family of proteins. To better understand the basis for the biophysical diversity of KCNQ1 channels, we here investigate the basis of KCNQ1 gating in the absence of β subunits using voltage-clamp fluorometry (VCF). In our previous study, we found the kinetics and voltage dependence of voltage-sensor movements are very similar to those of the channel gate, as if multiple voltage-sensor movements are not required to precede gate opening. Here, we have tested two different hypotheses to explain KCNQ1 gating: (i) KCNQ1 voltage sensors undergo a single concerted movement that leads to channel opening, or (ii) individual voltage-sensor movements lead to channel opening before all voltage sensors have moved. Here, we find that KCNQ1 voltage sensors move relatively independently, but that the channel can conduct before all voltage sensors have activated. We explore a KCNQ1 point mutation that causes some channels to transition to the open state even in the absence of voltage-sensor movement. To interpret these results, we adopt an allosteric gating scheme wherein KCNQ1 is able to transition to the open state after zero to four voltage-sensor movements. This model allows for widely varying gating behavior, depending on the relative strength of the opening transition, and suggests how KCNQ1 could be controlled by coassembly with different KCNE family members.     

Double electron–electron resonance reveals cAMP-induced conformational change in HCN channels

Binding of 3′,5′-cyclic adenosine monophosphate (cAMP) to hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels regulates their gating. cAMP binds to a conserved intracellular cyclic nucleotide-binding domain (CNBD) in the channel, increasing the rate and extent of activation of the channel and shifting activation to less hyperpolarized voltages. The structural mechanism underlying this regulation, however, is unknown. We used double electron–electron resonance (DEER) spectroscopy to directly map the conformational ensembles of the CNBD in the absence and presence of cAMP. Site-directed, double-cysteine mutants in a soluble CNBD fragment were spin-labeled, and interspin label distance distributions were determined using DEER. We found motions of up to 10 Å induced by the binding of cAMP. In addition, the distributions were narrower in the presence of cAMP. Continuous-wave electron paramagnetic resonance studies revealed changes in mobility associated with cAMP binding, indicating less conformational heterogeneity in the cAMP-bound state. From the measured DEER distributions, we constructed a coarse-grained elastic-network structural model of the cAMP-induced conformational transition. We find that binding of cAMP triggers a reorientation of several helices within the CNBD, including the C-helix closest to the cAMP-binding site. These results provide a basis for understanding how the binding of cAMP is coupled to channel opening in HCN and related channels.Ion channels are allosteric membrane proteins that open selective pores in response to various physiological stimuli, including binding of ligands and changes in transmembrane voltage (1). They are important for diverse physiological functions ranging from neurotransmission to muscle contraction. One such channel, the hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel, underlies the current (termed I_h, I_f, or I_q) produced in response to hyperpolarization of cardiac pacemaker cells and neurons (2). In the heart, HCN channels are responsible for pace-making activity and may have a role in the autonomic regulation of the heart rate (3–5). In the brain, HCN channels are involved in repetitive firing of neurons and dendritic integration (6–8). Despite the important physiological roles of HCN channels, the structure of the channels and molecular mechanism of their function are not completely understood.HCN channels are part of the voltage-gated K⁺ channel superfamily (9). Like other members of this family, they are tetramers, with each subunit having a voltage-sensor domain of four transmembrane helices (S1–S4) and a pore-lining domain consisting of two transmembrane helices separated by a reentrant loop (S5-P-S6; Fig. 1A). However, HCN channels contain two key specializations that make them unique among the voltage-gated ion channels: (i) They are activated by membrane hyperpolarization instead of depolarization, and (ii) they are regulated by the direct binding of cyclic nucleotides, like the ubiquitous second messenger cAMP, to a cytoplasmic domain in the carboxyl-terminal region of the channel. The direct binding of the agonist cAMP to HCN channels increases the rate and extent of activation and shifts the voltage dependence of activation to more depolarizing voltages.Open in a separate windowFig. 1.Study of conformational changes in HCN2 using DEER. (A, Upper) Putative transmembrane topology of HCN2 channels highlighting the voltage sensor domain (S1–S4) and the pore domain (S5–S6). Only two subunits are shown. (A, Lower) Crystal structure [Protein Data Bank (PDB) ID code 3ETQ] of the cysteine-free cytoplasmic carboxyl-terminal domain of HCN2. One subunit of the tetramer is shown in color. (B) Schematic diagram showing the distance change between two cysteine-attached MTSL spin labels in a protein upon cAMP binding. In this example, the two positions are closer in the presence of cAMP. (C) Raw DEER time traces for HCN2_cys-free V537C,A624C labeled with MTSL are shown in black in the absence or presence of cAMP, as indicated. The colored curves are distance-distribution fits to the data. The oscillation frequency is higher in the presence of cAMP, indicating that the two positions are closer together in the ligand-bound form.The crystal structure of the carboxyl-terminal region bound to cAMP has been solved for several HCN channels (10–14). The nearly identical structures consist of fourfold symmetrical tetramers predicted to connect directly to the S6 segments that form the ion-conducting pore (Fig. 1A). Each of the subunits contains two domains: the cyclic nucleotide-binding domain (CNBD) and the C-linker domain. The CNBD exhibits strong structural similarity to the CNBDs of other cyclic nucleotide-binding proteins, including cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac, and the Escherichia coli catabolite gene activator protein (CAP) (15–19). The CNBD consists of an eight-stranded antiparallel β-roll, followed by two α-helices (B-helix and C-helix). cAMP binds in the anticonformation between the β-roll and the C-helix. The C-linker is a unique domain found only in HCN channels and their close homologs, cyclic nucleotide-gated (CNG) channels, and KCNH family K⁺ channels (14, 20, 21). It is situated between the CNBD and membrane-spanning domains of the channel, and is the site of virtually all intersubunit interactions in the structure (Fig. 1A). The C-linker has been found to play a key role in coupling conformational changes in the CNBD to opening of the pore (9, 22, 23).The ligand-induced movement of the C-helix is widely thought to initiate the conformational changes that lead to opening of the channel pore, but the structural evidence in support of this hypothesis is equivocal (10, 24–29). The crystal structure of the HCN2 carboxyl-terminal region in the absence of ligand shows little difference from the cyclic nucleotide-bound structure (12). The only significant differences between the two structures are observed in the F′-helix of the C-linker and in the C-helix. The proximal half of the C-helix is in the same position in the cAMP-bound and unbound structures, whereas the distal half is missing from the apo structure, indicating that it is disordered or can access multiple conformations. In contrast, studies on the soluble carboxyl-terminal fragment using transition metal ion FRET (tmFRET) demonstrate a relatively large movement (∼5 Å) at the proximal end of the C-helix upon binding of cAMP (12). The tmFRET studies also indicate a smaller movement at the distal end of the C-helix and increased disorder in the C-helix in the absence of cyclic nucleotides (12, 26).In this study, we examined the cAMP-induced conformational transition in the CNBD of HCN2 using double electron–electron resonance (DEER) spectroscopy. DEER is a pulse electron paramagnetic resonance (EPR) method that can determine distances and resolve distance distributions between pairs of sites within proteins separated by about 15–80 Å (30–33). In a typical DEER experiment, two sites in a protein are mutated to cysteines and labeled with small magnetic spin labels (Fig. 1B). DEER measures the pair’s magnetic through-space coupling via excitation of one label and probing of the other with a series of short microwave pulses. This method yields an oscillating signal whose frequency falls off with the third power of the distance between the labels (Fig. 1C). Crucially, DEER measures full-distance distributions, rather than just an average distance, providing quantitative information on structural heterogeneity and variability that is not accessible from X-ray crystal structures or ensemble FRET experiments. Using DEER, we found that the binding of cAMP to the isolated C-linker/CNBD of HCN2 causes the C-helix to move substantially toward the β-roll and decreases the conformational heterogeneity of the protein. These observations are the first step in understanding the mechanisms of ligand gating of HCN channels and the activation of other CNBD-containing proteins.     

Clofarabine 5'-di and -triphosphates inhibit human ribonucleotide reductase by altering the quaternary structure of its large subunit

Human ribonucleotide reductases (hRNRs) catalyze the conversion of nucleotides to deoxynucleotides and are composed of α- and β-subunits that form active α_nβ_m (n, m = 2 or 6) complexes. α binds NDP substrates (CDP, UDP, ADP, and GDP, C site) as well as ATP and dNTPs (dATP, dGTP, TTP) allosteric effectors that control enzyme activity (A site) and substrate specificity (S site). Clofarabine (ClF), an adenosine analog, is used in the treatment of refractory leukemias. Its mode of cytotoxicity is thought to be associated in part with the triphosphate functioning as an allosteric inhibitor of hRNR. Studies on the mechanism of inhibition of hRNR by ClF di- and triphosphates (ClFDP and ClFTP) are presented. ClFTP is a reversible inhibitor (K_i = 40 nM) that rapidly inactivates hRNR. However, with time, 50% of the activity is recovered. D57N-α, a mutant with an altered A site, prevents inhibition by ClFTP, suggesting its A site binding. ClFDP is a slow-binding, reversible inhibitor (; t_1/2 = 23 min). CDP protects α from its inhibition. The altered off-rate of ClFDP from E•ClFDP^∗ by ClFTP (A site) or dGTP (S site) and its inhibition of D57N-α together implicate its C site binding. Size exclusion chromatography of hRNR or α alone with ClFDP or ClFTP, ± ATP or dGTP, reveals in each case that α forms a kinetically stable hexameric state. This is the first example of hexamerization of α induced by an NDP analog that reversibly binds at the active site.     

Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein

Light-oxygen-voltage (LOV) domains are blue light-activated signaling modules integral to a wide range of photosensory proteins. Upon illumination, LOV domains form internal protein-flavin adducts that generate conformational changes which control effector function. Here we advance our understanding of LOV regulation with structural, biophysical, and biochemical studies of EL222, a light-regulated DNA-binding protein. The dark-state crystal structure reveals interactions between the EL222 LOV and helix-turn-helix domains that we show inhibit DNA binding. Solution biophysical data indicate that illumination breaks these interactions, freeing the LOV and helix-turn-helix domains of each other. This conformational change has a key functional effect, allowing EL222 to bind DNA in a light-dependent manner. Our data reveal a conserved signaling mechanism among diverse LOV-containing proteins, where light-induced conformational changes trigger activation via a conserved interaction surface.     

Allosteric inhibition of complement function by a staphylococcal immune evasion protein

The complement system is a major target of immune evasion by Staphylococcus aureus. Although many evasion proteins have been described, little is known about their molecular mechanisms of action. Here we demonstrate that the extracellular fibrinogen-binding protein (Efb) from S. aureus acts as an allosteric inhibitor by inducing conformational changes in complement fragment C3b that propagate across several domains and influence functional regions far distant from the Efb binding site. Most notably, the inhibitor impaired the interaction of C3b with complement factor B and, consequently, formation of the active C3 convertase. As this enzyme complex is critical for both activation and amplification of the complement response, its allosteric inhibition likely represents a fundamental contribution to the overall immune evasion strategy of S. aureus.     

Selective Allosteric Antibodies to the Insulin Receptor for the Treatment of Hyperglycemic and Hypoglycemic Disorders

Many therapeutic monoclonal antibodies act as antagonists to receptors by targeting and blocking the natural ligand binding site (orthosteric site). In contrast, the use of antibodies to target receptors at allosteric sites (distinct from the orthosteric site) has not been extensively studied. This approach is especially important in metabolic diseases in which endogenous ligand levels are dysregulated. Herein, we review our investigations of 3 categories of human monoclonal antibodies that bind allosterically to the insulin receptor (INSR) and affect its activity: XMetA, XMetS and XMetD. XMetA directly activates the INSR either alone or in combination with insulin. XMetS, in contrast, does not directly activate the INSR but markedly enhances the receptor’s ability to bind insulin and potentiate insulin signaling. Both XMetA and XMetS are effective in controlling hyperglycemia in mouse models of diabetes. A third allosteric antibody, XMetD, is an inhibitor of INSR signaling. This antibody reverses insulin-induced hypoglycemia in a mouse model of hyperinsulinemia. These studies indicate, therefore, that allosteric antibodies to INSR can modulate its signaling and correct conditions of glucose dysregulation. These studies also raise the possibility that the use of allosteric antibodies can be expanded to other receptors for the treatment of metabolic disorders.     

Structural basis of activity and allosteric control of diguanylate cyclase

Recent discoveries suggest that a novel second messenger, bis-(3'-->5')-cyclic di-GMP (c-diGMP), is extensively used by bacteria to control multicellular behavior. Condensation of two GTP to the dinucleotide is catalyzed by the widely distributed diguanylate cyclase (DGC or GGDEF) domain that occurs in various combinations with sensory and/or regulatory modules. The crystal structure of the unorthodox response regulator PleD from Caulobacter crescentus, which consists of two CheY-like receiver domains and a DGC domain, has been solved in complex with the product c-diGMP. PleD forms a dimer with the CheY-like domains (the stem) mediating weak monomer-monomer interactions. The fold of the DGC domain is similar to adenylate cyclase, but the nucleotide-binding mode is substantially different. The guanine base is H-bonded to Asn-335 and Asp-344, whereas the ribosyl and alpha-phosphate moieties extend over the beta2-beta3-hairpin that carries the GGEEF signature motif. In the crystal, c-diGMP molecules are crosslinking active sites of adjacent dimers. It is inferred that, in solution, the two DGC domains of a dimer align in a two-fold symmetric way to catalyze c-diGMP synthesis. Two mutually intercalated c-diGMP molecules are found tightly bound at the stem-DGC interface. This allosteric site explains the observed noncompetitive product inhibition. We propose that product inhibition is due to domain immobilization and sets an upper limit for the concentration of this second messenger in the cell.     

State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels

Large-conductance voltage- and calcium-dependent potassium channels (BK, “Big K+”) are important controllers of cell excitability. In the BK channel, a large C-terminal intracellular region containing a “gating-ring” structure has been proposed to transduce Ca²⁺ binding into channel opening. Using patch-clamp fluorometry, we have investigated the calcium and voltage dependence of conformational changes of the gating-ring region of BK channels, while simultaneously monitoring channel conductance. Fluorescence resonance energy transfer (FRET) between fluorescent protein inserts indicates that Ca²⁺ binding produces structural changes of the gating ring that are much larger than those predicted by current X-ray crystal structures of isolated gating rings.     

Barium ions selectively activate BK channels via the Ca2+-bowl site

Activation of Ca(2+)-dependent BK channels is increased via binding of micromolar Ca(2+) to two distinct high-affinity sites per BK α-subunit. One site, termed the Ca(2+) bowl, is embedded within the second RCK domain (RCK2; regulator of conductance for potassium) of each α-subunit, while oxygen-containing residues in the first RCK domain (RCK1) have been linked to a separate Ca(2+) ligation site. Although both sites are activated by Ca(2+) and Sr(2+), Cd(2+) selectively favors activation via the RCK1 site. Divalent cations of larger ionic radius than Sr(2+) are thought to be ineffective at activating BK channels. Here we show that Ba(2+), better known as a blocker of K(+) channels, activates BK channels and that this effect arises exclusively from binding at the Ca(2+)-bowl site. Compared with previous estimates for Ca(2+) bowl-mediated activation by Ca(2+), the affinity of Ba(2+) to the Ca(2+) bowl is reduced about fivefold, and coupling of binding to activation is reduced from ～3.6 for Ca(2+) to about ～2.8 for Ba(2+). These results support the idea that ionic radius is an important determinant of selectivity differences among different divalent cations observed for each Ca(2+)-binding site.     

Structural identification of cation binding pockets in the plasma membrane proton pump

The activity of P-type plasma membrane H(+)-ATPases is modulated by H(+) and cations, with K(+) and Ca(2+) being of physiological relevance. Using X-ray crystallography, we have located the binding site for Rb(+) as a K(+) congener, and for Tb(3+) and Ho(3+) as Ca(2+) congeners. Rb(+) is found coordinated by a conserved aspartate residue in the phosphorylation domain. A single Tb(3+) ion is identified positioned in the nucleotide-binding domain in close vicinity to the bound nucleotide. Ho(3+) ions are coordinated at two distinct sites within the H(+)-ATPase: One site is at the interface of the nucleotide-binding and phosphorylation domains, and the other is in the transmembrane domain toward the extracellular side. The identified binding sites are suggested to represent binding pockets for regulatory cations and a H(+) binding site for protons leaving the pump molecule. This implicates Ho(3+) as a novel chemical tool for identification of proton binding sites.     

Differential global structural changes in the core particle of yeast and mouse proteasome induced by ligand binding

Two clusters of configurations of the main proteolytic subunit β5 were identified by principal component analysis of crystal structures of the yeast proteasome core particle (yCP). The apo-cluster encompasses unliganded species and complexes with nonpeptidic ligands, and the pep-cluster comprises complexes with peptidic ligands. The murine constitutive CP structures conform to the yeast system, with the apo-form settled in the apo-cluster and the PR-957 (a peptidic ligand) complex in the pep-cluster. In striking contrast, the murine immune CP classifies into the pep-cluster in both the apo and the PR-957–liganded species. The two clusters differ essentially by multiple small structural changes and a domain motion enabling enclosure of the peptidic ligand and formation of specific hydrogen bonds in the pep-cluster. The immune CP species is in optimal peptide binding configuration also in its apo form. This favors productive ligand binding and may help to explain the generally increased functional activity of the immunoproteasome. Molecular dynamics simulations of the representative murine species are consistent with the experimentally observed configurations. A comparison of all 28 subunits of the unliganded species with the peptidic liganded forms demonstrates a greatly enhanced plasticity of β5 and suggests specific signaling pathways to other subunits.Among the many factors involved in protein degradation through the ubiquitin-proteasome pathway, the core particle (CP) 20S proteasome plays the key role of the protease component. With the regulatory particle (RP), it forms a complex that selectively degrades ubiquitin-protein conjugates (1, 2). The CP in eukaryotes is a multisubunit complex composed of four stacked heptameric rings: two identical outer rings formed by seven different α subunits and two identical inner rings formed by seven different β subunits. The α_1–7β_1–7β_1–7α_1–7 organization defines a cylindrical structure (3). The α-rings control substrate entry into the lumen of the particle, where it is processed at the peptidolytic active centers, which are located at the inner walls of the β rings, specifically at subunits β1, β2, and β5. These active subunits are characterized by an N-terminal Thr residue. The other four β subunits have unprocessed N-terminal propeptides and are enzymatically inactive.All three active subunits share a common peptide hydrolyzing mechanism with two main steps (4): (i) the positioning of the substrate peptide in the active site by antiparallel alignment in between segments 47–49 and 21 of the active β subunits and (ii) peptide bond cleavage initiated by a nucleophilic attack of the hydroxyl group of the N-terminal Thr1 on the carbonyl carbon atom of the scissile peptide. Sequence diversity among β subunits endows them with distinctive structural features and different specificity pockets (S1, S2, S3, etc.) where the substrate side chains (P1, P2, P3, etc.) are bound (5). Consequently, the correlation of structural features of the S1 pockets with the distinctive cleavage products has led to the association of β1, β2, and β5 with caspase-like, trypsin-like, and chymotrypsin-like activities, respectively (6).The catalytically active subunits are substituted in immune cells of vertebrate organisms by the immune β-subunits β1i, β2i, and β5i as part of an adaptive immune response. These substitutions cause substantial functional differences between the constitutive (cCP) and immuno (iCP) species, reflected in higher yield of peptides that are recognized by the major histocompatibility complex (MHC) class I generated by iCP (7). Additionally, it has been observed that iCP achieves higher degradation rates than cCP, in both in vitro and cellular assays (8–13).Some sequence variations between the constitutive and immune subunits provide explanations to the observed catalytic differences. Most conspicuously, and first seen in the eukaryotic proteasome crystal structure from yeast (yCP) (3) and confirmed by the murine constitutive and immune CP structures (mcCP and miCP) (14), Arg45 of the β1 subunit, located at the base of the S1 pocket, is replaced by leucine in β1i, thereby causing a specific change of the electrostatic milieu, in line with the observed low postacidic activity of the iCP (15).Despite the high sequence similarity between β5 subunits of mcCP and miCP including identical active sites, a peptidic α-β-epoxyketone inhibitor, PR-957, showed higher affinity to iCP by one order of magnitude. The structural comparison of cCP and iCP in their apo and PR-957 liganded states suggested an explanation. On binding of PR-957, the cCP β5 backbone displays significant deformations, whereas the iCP β5 backbone remains unchanged. This observation, together with our experience in constructing β5 models for virtual screening purposes, prompted us to reinvestigate the vast amount of structural data for yCP by a procedure that facilitates discovery of global changes: principal component analysis (PCA).We focus our study on the β5 subunit, because β5 inactivation in yeast renders a lethal phenotype (16) and therefore β5 harbors an essential enzymatic activity, and because almost all crystallographically defined complexes are liganded at their β5 active site.Here we present a detailed investigation of the wealth of yeast and mouse proteasome ligand complex structures that led us to embark on structural comparisons beyond the immediate vicinity of the ligands to obtain a view of the global response of the core particle of yeast and mouse proteasome to complex formation. This study (i) is evidence of the structural plasticity of the β, specifically β5, subunits; (ii) offers perspectives for the analysis of the structure-function relationship of the CP; and (iii) provides an aid for the design and development of ligands as drugs for this intensively studied target for cancer and autoimmune diseases.     

Allosteric effects in the marginally stable von Hippel-Lindau tumor suppressor protein and allostery-based rescue mutant design

Many multifunctional tumor suppressor proteins have low stability, a property linked to cancer development. The von Hippel-Lindau tumor suppressor protein (pVHL) is one of these proteins. pVHL forms part of the E3 ubiquitin ligase complex that regulates the degradation of the hypoxia-inducible factor (HIF). Under native conditions, free pVHL is a molten globule, but it is stabilized in the E3 complex. By using molecular dynamics simulations, we observed that the interface between the two pVHL domains is the least stable region in unbound pVHL. We designed five stable mutants: one with a mutation at the interdomain interface and the others in the alpha- or beta-domains. Experimentally, type 2B pVHL disease mutant Y98N at the HIF binding site was shown to destabilize pVHL and decrease its binding affinity to HIF. Our simulations showed that the decrease in pVHL stability and binding affinity are allosterically regulated. The mutations designed to stabilize unbound wild-type pVHL, which are away from the elongin C and HIF binding sites, successfully stabilized the Y98N pVHL-elongin C complex and lowered the binding free energy of pVHL with HIF. Our results indicated both the enthalpic and dynamic allosteric components between the elongin C and HIF binding sites in pVHL, in the alpha- and beta-domains, respectively, mediated by the interdomain interface and linker. Drugs mimicking the allosteric effects of these mutants may rescue pVHL function in von Hippel-Lindau disease.     

Modulation of γ-secretase specificity using small molecule allosteric inhibitors

γ-Secretase cleaves multiple substrates within the transmembrane domain that include the amyloid precursor protein as well as the Notch family of receptors. These substrates are associated with Alzheimer disease and cancer. Despite extensive investigation of this protease, little is known regarding the regulation of γ-secretase specificity. To discover selective inhibitors for drug development and for probing the mechanisms of γ-secretase specificity, we screened chemical libraries and consequently developed a di-coumarin family of inhibitors that preferentially inhibit γ-secretase-mediated production of Aβ42 over other cleavage activities. These coumarin dimer-based compounds interact with γ-secretase by binding to an allosteric site. By developing a multiple photo-affinity probe approach, we demonstrate that this allosteric binding causes a conformational change within the active site of γ-secretase at the S2 and S1 sub-sites that leads to selective inhibition of Aβ42. In conclusion, by using these di-coumarin compounds, we reveal a mechanism by which γ-secretase specificity is regulated and provide insights into the molecular basis by which familial presenilin mutations may affect the active site and specificity of γ-secretase. Furthermore, this class of selective inhibitors provides the basis for development of Alzheimer disease therapeutic agents.