Domain Closure Research Articles

Coupled Control of Desensitization and Gating by the Ligand Binding Domain of Glutamate Receptors

The kinetics of ligand gated ion channels are tuned to permit diverse roles in cellular signaling. To follow high-frequency excitatory synaptic input, postsynaptic AMPA-type glutamate receptors must recover rapidly from desensitization. Chimeras between AMPA and the related kainate receptors demonstrate that the ligand binding domains alone control the lifetime of the desensitized state. Mutation of nonconserved amino acids in the lower lobe (domain 2) of the ligand binding domain conferred slow recovery from desensitization on AMPA receptors, and fast recovery on kainate receptors. Single-channel recordings and a correlation between the rate of deactivation and the rate of recovery across panels of mutant receptors revealed that domain 2 also controls ion channel gating. Our results demonstrate that the same mechanism that ensures fast recovery also sharpens the response of AMPA channels to synaptically released glutamate.

Re-evaluating the decoding principle

Following delivery of tRNA to the aminoacyl-tRNA binding site (A site) of the ribosome, a proofreading step ensures that the selected tRNA matches the mRNA codon of the A site. Previous studies suggested that the universally conserved nucleotides G530, A1492 and A1493 of 16S ribosomal RNA (rRNA) promote a conformational change (domain closure) on the 30S ribosomal subunit only when they detect the formation of Watson–Crick base pairs at the first two positions of the codon–anticodon helix. Here, Demeshkina et al. challenge this view showing that 30S domain closure also occurs in the presence of nucleotide mismatches at the codon–anticodon helix. Moreover, G530, A1492 and A1493 nucleotides of the 16S rRNA interact in a similar manner with mismatched base-pairs as with Watson–Crick base pairs. Thus, it seems that it is the formation of a tight decoding centre following 30S domain closure that destabilizesthe incorrect base pairs, leading to the release of near-cognate tRNA.

A new understanding of the decoding principle on the ribosome

During protein synthesis, the ribosome accurately selects transfer RNAs (tRNAs) in accordance with the messenger RNA (mRNA) triplet in the decoding centre. tRNA selection is initiated by elongation factor Tu, which delivers tRNA to the aminoacyl tRNA-binding site (A site) and hydrolyses GTP upon establishing codon-anticodon interactions in the decoding centre. At the following proofreading step the ribosome re-examines the tRNA and rejects it if it does not match the A codon. It was suggested that universally conserved G530, A1492 and A1493 of 16S ribosomal RNA, critical for tRNA binding in the A site, actively monitor cognate tRNA, and that recognition of the correct codon-anticodon duplex induces an overall ribosome conformational change (domain closure). Here we propose an integrated mechanism for decoding based on six X-ray structures of the 70S ribosome determined at 3.1-3.4 Å resolution, modelling cognate or near-cognate states of the decoding centre at the proofreading step. We show that the 30S subunit undergoes an identical domain closure upon binding of either cognate or near-cognate tRNA. This conformational change of the 30S subunit forms a decoding centre that constrains the mRNA in such a way that the first two nucleotides of the A codon are limited to form Watson-Crick base pairs. When U·G and G·U mismatches, generally considered to form wobble base pairs, are at the first or second codon-anticodon position, the decoding centre forces this pair to adopt the geometry close to that of a canonical C·G pair. This by itself, or with distortions in the codon-anticodon mini-helix and the anticodon loop, causes the near-cognate tRNA to dissociate from the ribosome.

Kainate induces various domain closures in AMPA and kainate receptors

Ionotropic glutamate receptors are key players in fast excitatory synaptic transmission within the central nervous system. These receptors have been divided into three subfamilies: the N-methyl-d-aspartic acid (NMDA), 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) and kainate receptors. Kainate has previously been crystallized with the ligand binding domain (LBD) of AMPA receptors (GluA2 and GluA4) and kainate receptors (GluK1 and GluK2). Here, we report the structures of the kainate receptor GluK3 LBD in complex with kainate and GluK1 LBD in complex with kainate in the absence of glycerol. Kainate introduces a conformational change in GluK3 LBD comparable to that of GluK2, but different from the conformational changes induced in GluA2 and GluK1. Compared to their domain closures in a glutamate bound state, GluA2 and GluK1 become more open and kainate induces a domain closure of 60% and 62%, respectively, relative to glutamate (100%). In GluK2 and GluK3 with kainate, the domain closure is 88% and 83%, respectively. In previously determined structures of GluK1 LBD in complex with kainate, glycerol is present in the binding site where it bridges interlobe residues and thus, might contribute to the large domain opening. However, the structure of GluK1 LBD with kainate in the absence of glycerol confirms that the observed domain closure is not an artifact of crystallization conditions. Comparison of the LBD structures with glutamate and kainate reveals that contacts are lost upon binding of kainate in the three kainate receptors, which is in contrast to the AMPA receptors where similar contacts are seen. It was revealed by patch clamp electrophysiology studies that kainate is a partial agonist at GluK1 with 36% efficacy compared to glutamate, which is in between the published efficacies of kainate at GluK2 and AMPA receptors. The ranking of efficacies seems to correlate with LBD domain closures.

Structural and Pharmacological Characterization of Phenylalanine‐Based AMPA Receptor Antagonists at Kainate Receptors

Continued efforts into the discovery of ligands that target ionotropic glutamate receptors (iGluRs) are important for studies of the physiological roles of the various iGluR subtypes as well as for the search for drugs that can be used in the treatment of diseases of the central nervous system. A new series of phenylalanine derivatives that target iGluRs was reported to bind AMPA receptors. Herein we report our studies of these compounds at the kainate receptors GluK1-3. Several compounds bind with micromolar affinity at GluK1 and GluK3, but do not bind GluK2. The crystal structure of the most potent compound in the ligand binding domain of GluK1 revealed different modes of binding to GluK1 and GluA2, due primarily to residues Ser741 (GluK1) and Met729 (GluA2). The compound was shown to be slightly more potent at GluK1 than at AMPA receptors and to induce a domain closure similar to that observed in GluK1 structures with partial agonists.

An Ionic Switch in the Ligand-Binding Domain of Non-NMDA Receptors

The ionotropic glutamate receptors (iGluRs), activated by the amino acid L-glutamate, account for the vast majority of excitatory neurotransmission in the central nervous system. Given their centrality to the nervous system, it is not surprising that these receptors have been linked to various neurological disorders including epilepsy, Alzheimer's and Parkinson's disease. Based on their sequence, pharmacological properties and function, iGluRs are classified primarily into three subtypes, namely amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate (KA), and N-methyl-D-aspartate (NMDA) receptors.Previously we have reported the identification of a potential ionic switch, or latch, located within the hinge region of non-NMDA receptors. Here we describe extensive molecular dynamics simulations to explore the conformational behaviour of the ionic switch and its influence on the closure or opening of the ligand-binding domain. The position of the switch appears to directly control the conformation of the ligand-binding domain. We discuss the results with respect to the general mechanism of how different ligands are able to induce the same mechanism of domain closure.

Interrogating Protein Conformational Mobility by Hydrogen Exchange Mass Spectrometry

It is often postulated that protein conformational mobility underlies enzyme activation, however our knowledge about how this occurs is still incomplete. To study this, we are measuring protein hydrogen/deuterium exchange by mass spectrometry (HX-MS) which reports internal motions of the folded state, where exchange predominantly occurs through low energy fluctuations in protein structure that enable transient solvent accessibility. Such fluctuations, represented by free energy differences (ααG) on the order of 1 kcal/mol, lead to changes in protein function even where structural differences are absent. HX-MS analysis of the mitogen-activated protein kinase, ERK2, reveals altered conformational mobility within localized regions of the enzyme upon catalytic activation. HX changes can be ascribed to enhanced backbone flexibility at the hinge between the conserved N- and C-terminal domains, predicting that interdomain closure may occur following ERK2 activation. This is corroborated by evidence that ERK2 binds nucleotide in two modes, where the active kinase adopts a closed conformation, whereas inactive ERK2 is constrained in an open conformation. Thus, phosphorylation of ERK2 releases constraints to interdomain closure needed for catalytic turnover, by regulating protein flexibility. Importantly, patterns of activation-induced HX differ between ERK2 and its paralog ERK1, despite their similarities in overall deuteration. Furthermore, ERK1 lacks constraints to domain closure prior to activation. Therefore, although MAP kinases are closely related with respect to primary sequence and tertiary structure, they utilize distinct mechanisms for dynamic control of enzyme function.

Structural Insight into Activation Mechanism of Toxoplasma gondii Nucleoside Triphosphate Diphosphohydrolases by Disulfide Reduction

The intracellular parasite Toxoplasma gondii produces two nucleoside triphosphate diphosphohydrolases (NTPDase1 and -3). These tetrameric, cysteine-rich enzymes require activation by reductive cleavage of a hitherto unknown disulfide bond. Despite a 97% sequence identity, both isozymes differ largely in their ability to hydrolyze ATP and ADP. Here, we present crystal structures of inactive NTPDase3 as an apo form and in complex with the product AMP to resolutions of 2.0 and 2.2 Å, respectively. We find that the enzyme is present in an open conformation that precludes productive substrate binding and catalysis. The cysteine bridge 258-268 is identified to be responsible for locking of activity. Crystal structures of constitutively active variants of NTPDase1 and -3 generated by mutation of Cys(258)-Cys(268) show that opening of the regulatory cysteine bridge induces a pronounced contraction of the whole tetramer. This is accompanied by a 12° domain closure motion resulting in the correct arrangement of all active site residues. A complex structure of activated NTPDase3 with a non-hydrolyzable ATP analog and the cofactor Mg(2+) to a resolution of 2.85 Å indicates that catalytic differences between the NTPDases are primarily dictated by differences in positioning of the adenine base caused by substitution of Arg(492) and Glu(493) in NTPDase1 by glycines in NTPDase3.

Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold

The joint substitution of three active-site residues in Escherichia colil-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 105-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, kcat′ for desulfination of l-cysteine sulfinate increased to 0.5s−1 (from 0.05s−1 in wild-type enzyme), whereas kcat′ for transamination of the same substrate was reduced from 510s−1 to 0.05s−1. Similarly, kcat′ for β-decarboxylation of l-aspartate increased from<0.0001s−1 to 0.07s−1, whereas kcat′ for transamination was reduced from 530s−1 to 0.13s−1. l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate form or liganded with a covalent coenzyme–substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme–substrate intermediates by bulk water.

Transmembrane Gate Movements in the Type II ATP-binding Cassette (ABC) Importer BtuCD-F during Nucleotide Cycle

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.

A New Phenylalanine Derivative Acts as an Antagonist at the AMPA Receptor GluA2 and Introduces Partial Domain Closure: Synthesis, Resolution, Pharmacology, and Crystal Structure

In order to map out molecular determinants for competitive blockade of AMPA receptor subtypes, a series of 2-carboxyethylphenylalanine derivatives has been synthesized and pharmacologically characterized in vitro. One compound in this series, (RS)-3h, showed micromolar affinity for GluA1(o) and GluA2(R)(o) receptors with an approximately 4-fold preference for GluA1/2 vs GluA3/4. In TEVC electrophysiological experiments (RS)-3h competitively antagonized GluA2(Q)(i) receptors. The X-ray structure of the active enantiomer (S)-3h in complex with GluA2-S1S2J showed a domain closure around 8°. Even though the nitro and the carboxyethyl groups of (S)-3h were both anchored to Tyr702 through a water H-bond network, these interactions only induced weak subtype selectivity. In spite of the fact that (S)-3h induced a domain closure close to that observed for partial agonists, it did not produce agonist responses at GluA2 receptors under nondesensitizing conditions. 2-Carboxyethylphenylalanine derivatives provide a new synthetic scaffold for the introduction of substituents that could lead to AMPA receptor subtype-selective ligands.

Essential role of the metal-ion in the IPM-assisted domain closure of 3-isopropylmalate dehydrogenase

X-ray structures of 3-isopropylmalate dehydrogenase (IPMDH) do not provide sufficient information on the role of the metal-ion in the metal–IPM assisted domain closure. Here solution studies were carried out to test its importance. Small-angle X-ray scattering (SAXS) experiments with the Thermus thermophilus enzyme (complexes with single substrates) have revealed only a very marginal (0–5%) extent of domain closure in the absence of the metal-ion. Only the metal–IPM complex, but neither the metal-ion nor the free IPM itself, is efficient in stabilizing the native protein conformation as confirmed by denaturation experiments with Escherichia coli IPMDH and by studies of the characteristic fluorescence resonance energy transfer (FRET) signal (from Trp to bound NADH) with both IPMDHs. A possible atomic level explanation of the metal-effect is given.

Binding site and interlobe interactions of the ionotropic glutamate receptor GluK3 ligand binding domain revealed by high resolution crystal structure in complex with (S)-glutamate

Ionotropic glutamate receptors (iGluRs) are involved in excitatory signal transmission throughout the central nervous system and their malfunction is associated with various health disorders. GluK3 is a subunit of iGluRs, belonging to the subfamily of kainate receptors (GluK1-5). Several crystal structures of GluK1 and GluK2 ligand binding domains have been determined in complex with agonists and antagonists. However, little is known about the molecular mechanisms underlying GluK3 ligand binding properties and no compounds displaying reasonable selectivity towards GluK3 are available today. Here, we present the first X-ray crystal structure of the ligand binding domain of GluK3 in complex with glutamate, determined to 1.6Å resolution. The structure reveals a conserved glutamate binding mode, characteristic for iGluRs, and a water molecule network in the glutamate binding site similar to that seen in GluK1. In GluK3, a slightly lower degree of domain closure around glutamate is observed compared to most other kainate receptor structures with glutamate. The volume of the GluK3 glutamate binding cavity was found to be of intermediate size between those of GluK1 and GluK2. The residues in GluK3 contributing to the subfamily differences in the binding sites are primarily: Thr520, Ala691, Asn722, Leu736 and Thr742. The GluK3 ligand binding domain seems to be less stabilized through interlobe interactions than GluK1 and this may contribute to the faster desensitization kinetics of GluK3.

Binding and Selectivity of the Marine Toxin Neodysiherbaine A and Its Synthetic Analogues to GluK1 and GluK2 Kainate Receptors

Dysiherbaine (DH) and neodysiherbaine A (NDH) selectively bind and activate two kainate-type ionotropic glutamate receptors, GluK1 and GluK2. The ligand-binding domains of human GluK1 and GluK2 were crystallized as bound forms with a series of DH analogues including DH, NDH, 8-deoxy-NDH, 9-deoxy-NDH and 8,9-dideoxy-NDH (MSVIII-19), isolated from natural sources or prepared by total synthesis. Since the DH analogues exhibit a wide range of binding affinities and agonist efficacies, it follows that the detailed analysis of crystal structure would provide us with a significant opportunity to elucidate structural factors responsible for selective binding and some aspects of gating efficacy. We found that differences in three amino acids (Thr503, Ser706 and Ser726 in GluK1 and Ala487, Asn690 and Thr710 in GluK2) in the ligand-binding pocket generate differences in the binding modes of NDH to GluK1 and GluK2. Furthermore, deletion of the C9 hydroxy group in NDH alters the ligand conformation such that it is no longer suited for binding to the GluK1 ligand-binding pocket. In GluK2, NDH pushes and rotates the side chain of Asn690 (substituted for Ser706 in GluK1) and disrupts an interdomain hydrogen bond with Glu409. The present data support the idea that receptor selectivities of DH analogues resulted from the differences in the binding modes of the ligands in GluK1/GluK2 and the steric repulsion of Asn690 in GluK2. All ligands, regardless of agonist efficacy, induced full domain closure. Consequently, ligand efficacy and domain closure did not directly coincide with DH analogues and the kainate receptors.

Importance of Domain Closure for the Autoactivation of ERK2

Extracellular signal-regulated kinases 1 and 2 (ERK1 and -2, respectively) play a critical role in regulating cell division and have been implicated in cancer. In addition to activation by MAPK/ERK kinases 1 and 2 (MEK1 and -2, respectively), certain mutants of ERK2 can be activated by autophosphorylation. To identify the mechanism of autoactivation, we have performed a series of molecular dynamics simulations of ERK1 and -2 in various stages of activation as well as the constitutively active Q103A, I84A, L73P, and R65S ERK2 mutants. Our simulations indicate the importance of domain closure for autoactivation and activity regulation, with that event occurring prior to folding of the activation lip and of loop L16. Results indicate that the second phosphorylation event, that of T183, disrupts hydrogen bonding involving D334, thereby allowing the kinase to lock into the active conformation. On the basis of the simulations, three predictions were made. G83A was suggested to impede activation; K162M was suggested to perturb the interface between the N- and C-domains leading to activation, and Q64C was hypothesized to stop folding of loop L16, thereby perturbing the homodimerization interface. Functional analysis of the mutants validated the predictions concerning the G83A and Q64C mutants. The K162M mutant did not autoactivate as predicted, however, which may be due to the location of the residue on the protein surface near the ED substrate docking domain.

Investigating of interactions between statin-based cholesterol lowering drugs with p-glycoprotein membrane protein by molecular modeling

In this study, the interaction of P-glycoprotein (P-gp) with lactone form of cholesterol lowering atorvastatin and linear peptides including ALLM (N-acetyl-leu-leu-met-al) and AFMRF (N-acetyl-phemet-arg-phe-al) were investigated by using molecular dynamics simulation. P-gp is a multidrug resistance protein which expels the toxic compounds out of the cell and in this way protects the cell from the harmful effect of these compounds. First, P-gp was integrated into 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC) lipid bilayer and solvated to investigate these interactions. Then Mg-ATP molecules, which supply the required energy for transportation, were docked into the nucleotide binding domains of the protein. After the preparation of the P-gp integrated lipid membrane system, simulations were run for each substrate. As a result, while no interaction was detected between the linear peptides and P-gp, lactone form of atorvastatin was found to have an interaction with the protein, leading to an asymmetrical closure of nucleotide binding domains. When the ability of P-gp to transport multiple substrates were taken into account, it could be said that single small peptides could not trigger the protein activity needed for transportation. The authors would like to acknowledged the grant from ITU-BAP.

Evaluation of natural and nitramine binding energies to 3-D models of the S1S2 domains in the N-methyl-D-aspartate receptor

Overactivation of the N-methyl-D-aspartate receptor (NMDAR) in postsynaptic neurons leads to glutamate-related excitotoxicity in the central nervous system of mammals. We have built 3-D models of each domain for the universal screening of potential toxicants and their binding mechanisms. Our docking results show that the calculated pK (i) values of glycine and L: -glutamate significantly increase (>1) when the NR1 and NR2A S1S2 domains are closing, respectively. Inversely, D: -cycloserine (DCS) and 5,7-dichlorokynurenic acid (5,7-DCKA) do not show such a dependence on domain closure. Replica exchange molecular dynamics (REMD) confirmed 5 different conformational states of the S1S2 domain along the 308.2 K temperature trajectory. Analysis of residue fluctuations during this temperature trajectory showed that residues in loop 1, loop 2, the amino terminal domain (ATD), and the area linked to ion channel α-helices are involved in this movement. This further implicates the notion that efficacious ligands act through S1S2 lobe movement which can culminate in the opening or closing of the ion channel. We further tested this by docking hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX) to the S1S2 domain. Our results predict that these nitramines are not efficacious and thus do not produce excitoxicity when they bind to the S1S2 domain of the NMDAR.

Arg149 Is Involved in Switching the Low Affinity, Open State of the Binding Protein AfProX into Its High Affinity, Closed State

The substrate binding protein AfProX from the Archaeoglobus fulgidus ProU ATP binding cassette transporter is highly selective for the compatible solutes glycine betaine (GB) and proline betaine, which confer thermoprotection to this hyperthermophilic archaeon. A detailed mutational analysis of the substrate binding site revealed the contribution of individual amino acids for ligand binding. Replacement of Arg149 by an Ala residue displayed the largest impact on substrate binding. The structure of a mutant AfProX protein (substitution of Tyr111 with Ala) in complex with GB was solved in the open liganded conformation to gain further insight into ligand binding. In this crystal structure, GB is bound differently compared to the GB closed liganded structure of the wild-type AfProX protein. We found that a network of amino acid side chains communicates the presence of GB toward Arg149, which increases ligand affinity and induces domain closure of AfProX. These results were corroborated by molecular dynamics studies and support the view that Arg149 finalizes the high-affinity state of the AfProX substrate binding protein.

A Spring-loaded Release Mechanism Regulates Domain Movement and Catalysis in Phosphoglycerate Kinase

Phosphoglycerate kinase (PGK) is the enzyme responsible for the first ATP-generating step of glycolysis and has been implicated extensively in oncogenesis and its development. Solution small angle x-ray scattering (SAXS) data, in combination with crystal structures of the enzyme in complex with substrate and product analogues, reveal a new conformation for the resting state of the enzyme and demonstrate the role of substrate binding in the preparation of the enzyme for domain closure. Comparison of the x-ray scattering curves of the enzyme in different states with crystal structures has allowed the complete reaction cycle to be resolved both structurally and temporally. The enzyme appears to spend most of its time in a fully open conformation with short periods of closure and catalysis, thereby allowing the rapid diffusion of substrates and products in and out of the binding sites. Analysis of the open apoenzyme structure, defined through deformable elastic network refinement against the SAXS data, suggests that interactions in a mostly buried hydrophobic region may favor the open conformation. This patch is exposed on domain closure, making the open conformation more thermodynamically stable. Ionic interactions act to maintain the closed conformation to allow catalysis. The short time PGK spends in the closed conformation and its strong tendency to rest in an open conformation imply a spring-loaded release mechanism to regulate domain movement, catalysis, and efficient product release.

Vortex-antivortex creation and annihilation on CoFeB crosstie patterns

The creation and annihilation of vortex and antivortex pairs in crosstie walls on CoFeB islands were investigated using magnetic force microscopy (MFM) in the presence of applied field. The first vortex-antivortex pair (V-AV) is nucleated in a 4-domain Landau pattern from a section of a 180° degree Néel wall as a consequence of the formation of an end domain closure pattern. The V-AV pair creation satisfies the condition for the closure domain vortices to have the same chiralities. Pair annihilation occurs in two steps: a rapid advance of the vortices toward the more or less fixed antivortices followed by a deceleration caused by the decrease in local susceptibility. There is a spread in the annihilation fields that does not depend upon the core polarity but is determined by energy minimization.