Gideon Schreiber

Energetics of protein-protein interactions: Analysis ofthe Barnase-Barstar interface by single mutations and double mutant cycles

The interaction of barnase, an extracellular RNase of Bacillus amylolique-faciens, with its intracellular inhibitor barstar is a suitable paradigm for protein-protein interactions, since the structures of both the free and the complexed proteins are available at high resolution. The contributions of residues from both proteins to the energetics of kinetics and thermodynamics of binding were measured by double mutant cycle analysis. Such cycles reveal whether the contributions from a pair of residues are additive, or the effects of mutations are coupled. The aim of the study was to determine which of the interactions are co-operative. Double mutant cycles were constructed between a subset of five barnase and seven barstar residues, which were shown by structural and mutagenesis studies to be important in stabilising the complex. The coupling energy between two residues was found to decrease with the distance between them. Generally, residues separated by less than 7 A interact co-operatively. At greater separations, the effects of mutation are additive, and the energetics of the interactions are independent of each other. The highest coupling energies are found between pairs of charged residues (1.6 to 7 kcal mol-1). Three of the six most important interactions detected by double mutant cycle analysis (with coupling energies of more than 3.0 kcal mol-1) had not been noted previously from examination of the crystal structure. The effects of mutation on the kinetics of association are all additive, apart from charged residues located at distances of up to 10 A apart, which are co-operative. This can be explained by the fact that the transition state for association occurs before most interactions are formed.

Fundamental Aspects of Protein-Protein Association Kinetics

The structure of a protein complex, together with information about its affinity and other thermodynamic characteristics, provide a “frozen” view of the complex. This picture ignores the kinetic nature of protein-protein association and dissociation, which are of major biological and biophysical interest. This review focuses on recent advances in deciphering the kinetic pathway of protein complex formation, the nature of the pre-complex formed through diffusion (which we have termed the “transient complex”1), the transition state, and other intermediates (such as the so-called encounter complex) along the association pathway. Protein-protein association is at the center of diverse biological processes ranging from enzyme catalysis/inhibition to regulation of immune response by cytokines. The association rates often play a critical role in such processes, as in situations where speed is of essence.2 For example, the purple cone snail and other venomous animals capture prey with remarkable efficiency and speed by releasing toxins that rapidly bind to ion channels;3 the green mamba achieves a similar feat by targeting acetylcholinesterase (AChE), an enzyme essential for the integrity of neural transmission.4 Bacteria such as Escherichia coli and Bacillus amyloliquefaciens excrete nucleases as weapons against competitors or predators. Defense of the producing cells from damage to their own DNA or RNA by such nucleases requires rapid association with cognate inhibitors.5,6 Indeed, in the last example rapid association is such a priority that the inhibitor barstar has a cluster of acidic residues that facilitate association with the nuclease barnase, even though the clustered charges reduce folding stability.7 In the ruminant gut, RNase A is required for degrading accumulated RNA; potential toxicity of leaked nuclease is prevented by rapid association with a ribonuclease inhibitor.8,9 Reorganization of the actin cytoskeleton provides yet another illustration of the importance of rapid protein association. Reorganization is attained through actin polymerization, which is nucleated by the Arp2/3 complex. The latter is activated by the Wiskott-Aldrich Syndrome protein (WASp), which in turn is released from the auto-inhibited state by the Rho GTPase Cdc42.10 As actin polymerization is initiated with a nucleation process, the speed of upstream signaling has a critical impact on the rate of polymer formation. It is thus not surprising that high association rate constants have been observed between partners along the signaling pathway.11,12 The high association rate constant between Cdc42 and WASp has been found to be essential for the latter to stimulate actin polymerization, as another Rho GTPase sharing 70% sequence identity, TC10, with an identical dissociation rate constant but a 1000-fold lower association rate constant, failed to stimulate actin polymerization.11 The failure to stimulate actin polymerization in patients carrying mutant WAS genes is the root cause of the Wiskott-Aldrich Syndrome. Several other compelling arguments can be made for the biological roles of rapid protein association.13 (a) Fast association may enhance binding affinity. High affinity can also be achieved through slow dissociation; however, for proteins involved in signaling, slow dissociation is not an option, since it implies a long-lasting bound state, which effectively corresponds to a permanent off- or on-switch. A good example for this is the binding of Ras to its natural affector Raf. This protein dissociates within a fraction of a second, but maintains an affinity in the nM range through fast association. Moreover, the difference between the natural effector, Raf, and the non-natural effector, Ral, lies in their rates of association with Ras.14 Therefore, even if not for a direct reason (such as in stimulation of actin polymerization), the affinity requirement alone may call for fast association. (b) Enzyme-substrate binding is a determining factor for the overall turnover rate and becomes the rate-limiting step for catalytically “perfect” enzymes. Substrate-binding rate constants of such enzymes reach 108 M−1s−1 and beyond, as found for the ribotoxin restrictocin and RNase A.15,16 (c) When several proteins compete for the same receptor or when one protein is faced with alternative pathways, kinetic control, not thermodynamic control, dominates in many cases; this is especially true when dissociation is slow. For example, during protein synthesis cognate and noncognate aminoacyl-tRNA synthetases can potentially compete for the same tRNA. As an additional example, consider newly synthesized proteins, which potentially face aggregation if not isolated by a chaperone. From the point of view of kinetic control, it is easy to see why rapid binding of denatured proteins to the chaperonin GroEL has been observed.17 (d) Differences in binding rate between related proteins may serve as an additional mechanism for specificity, as can be suggested for Rho GTPases Cdc42 and TC10 and for Ras effectors Raf and Ral. The examples and arguments presented above suggest that rapid binding is as important as high affinity in the proper functioning of proteins. It is now increasingly recognized that proteins function in the context of multi-component complexes. Manipulating association rate constants of various components presents unique opportunities for the control of protein functions. Many interactions between proteins are also targeted for drug development; in designing such drugs, both high affinity and rapid binding should be taken into consideration. 1.1. Overview of Protein Association Kinetics The observed rate constants of protein association span a wide range, from 109 M−1s−1 (Figure 1). In comprehending these values, a basic fact is that, for two proteins to recognize each other, their interfaces have to be oriented with high specificity. A relative rotation of as little as a few degrees or a relative translation by a few Angstroms is sufficient to break all specific interactions between the two proteins.18 The rate of association of a protein complex is limited by diffusion and geometric constraints of the binding sites, and may be further reduced by subsequent chemical processes.19 Figure 1 The wide spectrum of association rate constants. The red vertical line marks the start of the diffusion-controlled regime. The shaded range marks the absence of long-range forces. Adapted with permission from Ref. 1. Copyright 2008 Wiley Interscience.. ... To better understand the kinetics of association of two proteins (A and B), it is useful to consider the process as going through an intermediate state (A*B), in which the two proteins have near-native separations and orientations.1,20–23 We refer to this intermediate state as the transient complex,1,20 noting that is sometimes also termed the encounter complex.24 A more detailed discussion of terminology, as well as the specification of the ensemble of configurations making up the transient complex is provided in Section 3. From this ensemble, conformational rearrangement can lead to the native complex (C). Accordingly we have the kinetic scheme A+B⇄k−DkDA∗B→kcC (1) While the first step of this scheme depends on relative diffusion between the protein molecules, the second step is akin to an intramolecular chemical reaction, and can therefore be described by the classical transition-state theory25 (with the transition state located at the top of the free energy barrier separating A*B from C26) or by Kramers’ theory.27 The latter theory accounts for barrier recrossing and models motion along the reaction coordinate as diffusive. The overall rate constant of association is ka=kDkck−D+kc (2) which is bounded by the diffusion-controlled rate constant, kD, for reaching the transient complex. This limit is reached when conformational rearrangement is fast relative to the dissociation of the transient complex (i.e., kc ≫ k−D), leading to

Rational design of faster associating and tighter binding protein complexes

A protein design strategy was developed to specifically enhance the rate of association (kon) between a pair of proteins without affecting the rate of dissociation (koff). The method is based on increasing the electrostatic attraction between the proteins by incorporating charged residues in the vicinity of the binding interface. The contribution of mutations towards the rate of association was calculated using a newly developed computer algorithm, which predicted accurately the rate of association of mutant protein complexes relative to the wild type. Using this design strategy, the rate of association and the affinity between TEM1 β-lactamase and its protein inhibitor BLIP was enhanced 250-fold, while the dissociation rate constant was unchanged. The results emphasize that long range electrostatic forces specifically alter kon, but do not effect koff. The design strategy presented here is applicable for increasing rates of association and affinities of protein complexes in general.

Type I interferon responses in rhesus macaques prevent SIV infection and slow disease progression

Inflammation in HIV infection is predictive of non-AIDS morbidity and death, higher set point plasma virus load and virus acquisition; thus, therapeutic agents are in development to reduce its causes and consequences. However, inflammation may simultaneously confer both detrimental and beneficial effects. This dichotomy is particularly applicable to type I interferons (IFN-I) which, while contributing to innate control of infection, also provide target cells for the virus during acute infection, impair CD4 T-cell recovery, and are associated with disease progression. Here we manipulated IFN-I signalling in rhesus macaques (Macaca mulatta) during simian immunodeficiency virus (SIV) transmission and acute infection with two complementary in vivo interventions. We show that blockade of the IFN-I receptor caused reduced antiviral gene expression, increased SIV reservoir size and accelerated CD4 T-cell depletion with progression to AIDS despite decreased T-cell activation. In contrast, IFN-α2a administration initially upregulated expression of antiviral genes and prevented systemic infection. However, continued IFN-α2a treatment induced IFN-I desensitization and decreased antiviral gene expression, enabling infection with increased SIV reservoir size and accelerated CD4 T-cell loss. Thus, the timing of IFN-induced innate responses in acute SIV infection profoundly affects overall disease course and outweighs the detrimental consequences of increased immune activation. Yet, the clinical consequences of manipulation of IFN signalling are difficult to predict in vivo and therapeutic interventions in human studies should be approached with caution.

Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions

Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science.

Assessing computational methods for predicting protein stability upon mutation: good on average but not in the details

Methods for protein modeling and design advanced rapidly in recent years. At the heart of these computational methods is an energy function that calculates the free energy of the system. Many of these functions were also developed to estimate the consequence of mutation on protein stability or binding affinity. In the current study, we chose six different methods that were previously reported as being able to predict the change in protein stability (DeltaDeltaG) upon mutation: CC/PBSA, EGAD, FoldX, I-Mutant2.0, Rosetta and Hunter. We evaluated their performance on a large set of 2156 single mutations, avoiding for each program the mutations used for training. The correlation coefficients between experimental and predicted DeltaDeltaG values were in the range of 0.59 for the best and 0.26 for the worst performing method. All the tested computational methods showed a correct trend in their predictions, but failed in providing the precise values. This is not due to lack in precision of the experimental data, which showed a correlation coefficient of 0.86 between different measurements. Combining the methods did not significantly improve prediction accuracy compared to a single method. These results suggest that there is still room for improvement, which is crucial if we want forcefields to perform better in their various tasks.

The Receptor of the Type I Interferon Family

All type I IFNs act through a single cell surface receptor composed of the IFNAR1 and IFNAR2 subunits and two associated cytoplasmic tyrosine kinases of the Janus family, Tyk2 and Jak1. A central issue in type I IFN biology is to understand how a multitude of subtypes can generate similar signaling outputs but also govern specific cellular responses. This review summarizes results from the last decade that contributed to our current state of knowledge of IFN-receptor complex structure and assembly.

Inquiring into the Differential Action of Interferons (IFNs): an IFN-α2 Mutant with Enhanced Affinity to IFNAR1 Is Functionally Similar to IFN-β

ABSTRACT Alpha and beta interferons (IFN-α and IFN-β) are multifunctional cytokines that exhibit differential activities through a common receptor composed of the subunits IFNAR1 and IFNAR2. Here we combined biophysical and functional studies to explore the mechanism that allows the alpha and beta IFNs to act differentially. For this purpose, we have engineered an IFN-α2 triple mutant termed the HEQ mutant that mimics the biological properties of IFN-β. Compared to wild-type (wt) IFN-α2, the HEQ mutant confers a 30-fold higher binding affinity towards IFNAR1, comparable to that measured for IFN-β, resulting in a much higher stability of the ternary complex as measured on model membranes. The HEQ mutant, like IFN-β, promotes a differentially higher antiproliferative effect than antiviral activity. Both bring on a down-regulation of the IFNAR2 receptor upon induction, confirming an increased ternary complex stability of the plasma membrane. Oligonucleotide microarray experiments showed similar gene transcription profiles induced by the HEQ mutant and IFN-β and higher levels of gene induction or repression than those for wt IFN-α2. Thus, we show that the differential activities of IFN-β are directly related to the binding affinity for IFNAR1. Conservation of the residues mutated in the HEQ mutant within IFN-α subtypes suggests that IFN-α has evolved to bind IFNAR1 weakly, apparently to sustain differential levels of biological activities compared to those induced by IFN-β.

Protein Binding Specificity versus Promiscuity

Interactions between macromolecules in general, and between proteins in particular, are essential for any life process. Examples include transfer of information, inhibition or activation of function, molecular recognition as in the immune system, assembly of macromolecular structures and molecular machines, and more. Proteins interact with affinities ranging from millimolar to femtomolar and, because affinity determines the concentration required to obtain 50% binding, the amount of different complexes formed is very much related to local concentrations. Although the concentration of a specific binding partner is usually quite low in the cell (nanomolar to micromolar), the total concentration of other macromolecules is very high, allowing weak and non-specific interactions to play important roles. In this review we address the question of binding specificity, that is, how do some proteins maintain monogamous relations while others are clearly polygamous. We examine recent work that addresses the molecular and structural basis for specificity versus promiscuity. We show through examples how multiple solutions exist to achieve binding via similar interfaces and how protein specificity can be tuned using both positive and negative selection (specificity by demand). Binding of a protein to numerous partners can be promoted through variation in which residues are used for binding, conformational plasticity and/or post-translational modification. Natively unstructured regions represent the extreme case in which structure is obtained only upon binding. Many natively unstructured proteins serve as hubs in protein-protein interaction networks and such promiscuity can be of functional importance in biology.

Community-wide assessment of protein-interface modeling suggests improvements to design methodology

The CAPRI (Critical Assessment of Predicted Interactions) and CASP (Critical Assessment of protein Structure Prediction) experiments have demonstrated the power of community-wide tests of methodology in assessing the current state of the art and spurring progress in the very challenging areas of protein docking and structure prediction. We sought to bring the power of community-wide experiments to bear on a very challenging protein design problem that provides a complementary but equally fundamental test of current understanding of protein-binding thermodynamics. We have generated a number of designed protein-protein interfaces with very favorable computed binding energies but which do not appear to be formed in experiments, suggesting that there may be important physical chemistry missing in the energy calculations. A total of 28 research groups took up the challenge of determining what is missing: we provided structures of 87 designed complexes and 120 naturally occurring complexes and asked participants to identify energetic contributions and/or structural features that distinguish between the two sets. The community found that electrostatics and solvation terms partially distinguish the designs from the natural complexes, largely due to the nonpolar character of the designed interactions. Beyond this polarity difference, the community found that the designed binding surfaces were, on average, structurally less embedded in the designed monomers, suggesting that backbone conformational rigidity at the designed surface is important for realization of the designed function. These results can be used to improve computational design strategies, but there is still much to be learned; for example, one designed complex, which does form in experiments, was classified by all metrics as a nonbinder.