Processivity is a kinetic phenomenon widespread among enzymes that act on polymeric substrates, such as DNA, RNA, polysaccharides, and proteins [1
]. Once committed, processive enzymes engage in multiple rounds of modification instead of releasing their substrate after modifying it once. Served by different sliding mechanism(s), very effective enzymatic modifiers arose in evolution that can carry out hundreds or thousands of elementary steps upon a single engagement with the substrate [1
]. Processivity occurs in: (i) synthesis (e.g., DNA by DNA polymerase [2
], RNA by RNA polymerase, and protein by the ribosome [3
]); (ii) degradation (e.g., DNA by DNAse [4
], RNA by RNAse [5
], polysaccharides by glycohydrolases [6
] or proteins by the proteasome [7
]); (iii) structural modification (e.g., DNA by helicase [9
]); (iv) chemical modification (e.g., ubiquitination of proteins by ubiquitin ligases [10
]); or (v) cargo transport (e.g., movement by mechanochemical motors kinesin, dynein and myosin [12
] along actin and tubulin tracks).
A compilation of domain-linker-domain (DLD)-type monomeric processive enzymes is taken from the comprehensive list given in Supplementary Table S1
. Important parameters including the length of predicted disordered linker, mean linker length of orthologous proteins (see Table S2
for species), κ value describing charge distribution, and the level of processivity (such as the length of processive move, the number of steps taken or the number of elementary substrate units covered, if determined at all), are given.
Given the extreme diversity of substrates upon which these processive enzymes act and also the variability of the chemical/mechanochemical changes they make, it is of little surprise that the molecular details of processivity are rather diverse, yet they are based on combinations of two basic designs principles. The classic and amply studied mechanism relies on structural confinement by circular/cylindrical or asymmetric binding domains or subunits of the enzymes. The former occurs, for example, when the PCNA subunit of DNA polymerase encircles the template DNA (Figure 1
A) to ensure that the enzyme adds a practically unlimited number of nucleotides [16
] to the growing DNA polymer. A closely related solution is used by HIV reverse transcriptase [18
], which has an asymmetric binding domain that strongly favors sliding along the RNA substrate over dissociating from it (Figure 1
B). A completely different mechanism has evolved in mechanochemical motors, such as kinesin and dynein, which move along polymeric protein tracks of tubulin [15
]. These dimeric proteins have long coiled-coil stalks and ATPase binding domains, which undergo conformational changes that result in a strong preference for rebinding following dissociation due to a proximity effect, i.e., spatial confinement (Figure 1
C). The region connecting the dimerization domain with the binding domain may even undergo transitions between ordered and disordered states [19
]. The latter class of processive motors suggests that the presence of two binding elements (motifs or domains) connected by long, conformationally adaptable/flexible linker region(s) appears to be a key element of processivity, which combines deterministic and probabilistic elements of binding [20
Here we generalize this concept by observing and analyzing that proteins in which binding domains are connected by a disordered linker may show probabilistic bias for re-binding over dissociation from their substrate, due to which they possess processive capacity. As structural disorder of proteins (intrinsically disordered protein/region, IDP/IDR) is widespread in eukaryotic proteomes [21
], this may be a frequently applied mechanism. IDPs/IDRs often engage in protein-protein interactions [23
] but their function may also directly stem from the disordered state, termed entropic-chain functions [25
]. Binding and entropic-chain functions can actually be combined because often part of the IDP remains disordered even in the bound state, a phenomenon termed fuzziness [26
]. Of particular relevance to the observed processivity is that binding motifs embedded in disordered regions, due to the arising “proximity effect” or “optimal effective concentration” around binding sites, may feature facilitated binding, which is central to the concepts of: (i) acceleration of binding by “fly casting” [27
], (ii) reduction of binding dimensionality by the “monkey-bar” mechanism [28
], and (iii) “ultrasensitive” binding by repetitive binding motifs in signaling proteins [29
By statistical-physical modeling and bioinformatics analysis we show that this kinetic proximity effect is also a widespread inherent property of many monomeric processive enzymes that are capable of multiple rounds of modification of their polymeric substrate. These enzymes, such as a variety of glycohydrolases (e.g., cellulases) [6
], Ribonuclease H1 (RNAse-H1) [5
] and matrix metalloproteinase-9 (MMP-9) [33
], need no ATP energy for processivity, which makes it a robust and widespread mechanism in the proteome. Here we have selected 12 such monomeric (ATP-independent) processive enzymes from the literature and provide a comprehensive analysis of their physical and structural properties. We show that once engaged with their substrate, their structural organization kinetically biases binding of their free binding domain over dissociation of both its domains, resulting in multiple successive binding events without ever fully releasing the polymeric partner (Figure 1
D). We suggest that this type of processivity represents a unique type of “entropic chain” function enabled by the structural disorder of their linker region [25
], which may be a general mechanism that arises in a broad range of biological contexts.
Processivity is a basic device of enzymes working on (generating, modifying or moving along) polymeric substrates [1
]. By its very molecular logic, it increases cellular economy by limiting the production of metabolic by-products and the dissipation of energy, and it enables large-scale molecular changes to occur, thus it is at the heart of many key cellular processes. Due to the all-or-none character of the operation of processive enzymes, however, there have to be very precise and highly controlled cellular mechanisms for turning them on.
As outlined, there are diverse molecular mechanisms underlying processivity, falling into two general categories, structural confinement by well-folded binding elements and spatial confinement by independent binding elements connected through a linker region. This latter mechanism is apparent in dimeric mechanochemical motors and also in monomeric enzymes. The importance of the general kinetic consequence of processivity can be deduced from its convergent appearance in many independent systems. Whereas its mechanistic underpinning is rather well understood in the case of enzymes that rely on structural confinement and is also analyzed rather extensively in the case of mechanochemical motors, it has so far been largely overlooked in the case of monomeric enzymes.
The typical design of such enzymes is embodied by certain bacterial cellulases, which have a modular structure that combines a large CD linked to a smaller CBM by an intrinsically disordered linker [39
] that enables a continuum of conformations. A similar feature has been suggested for the matrix metalloproteinase MMP-9 [33
], which progressively degrades polymeric components of the extracellular matrix, such as collagen. This enzyme also has a modular structure, with an N-terminal unit of a catalytic domain and three fibronectin type II exosite modules, connected by a 54-residues long linker to a C-terminal hemopexin C domain. SAXS and AFM demonstrated that it can assume multiple conformations and that it can crawl in an inchworm-like manner along its substrate [57
]. A similar architecture has been suggested and/or theoretically modelled in the case of glycohydrolases, such as Cel7A [58
], cellobiohydrolase I [59
] and chitinases [60
]. The importance of this arrangement is underscored by cellobiohydrolase I, in which the deletion of the linker dramatically reduces the rate of crystalline cellulose degradation [32
] and also other glycoside hydrolases, in which the removal of the carbohydrate-binding module results in a significant decrease in their activity [6
], without directly affecting their catalytic domain. Apparently, the unifying feature of all these examples is the structural disorder of their linkers, which ensures a high local concentration and relatively restricted conformational search of binding domains around their binding sites.
Here, we used statistical-kinetic modelling of such systems that this structural arrangement can endow such an enzyme with the capacity of processive movements along a polymeric substrate of spatially repeating binding sites. We characterized these enzymes by the time of (re)binding as a function of linker length, and found that within a certain length range, they have a preference for binding over dissociation, i.e., they show processive kinetic behavior. Geometric features of the domains, direct binding of the linker with the domains themselves and PTMs of the linkers all influence binding kinetics and may thus serve as points of regulatory input. This might be of no negligible importance, as the processive chain of events past the point of activation appears uncontrolled, which may have dire consequences. A proper regulatory input halting the reaction may be a remedy under some circumstances, as suggested by frequent PTMs of processive linkers (Table 2
) and their regulated binding to the flanking domains, as shown for MMP-9, for example [33
These theoretical observations have general relevance and are supported by a collection of 12 such enzymes that all have highly disordered linkers. Notably, despite rapid evolution and sequence variability of IDPs/IDRs in general, and disordered linker regions in particular, the length and flexibility of linkers in the processive enzymes is conserved. Quantitative modelling of the cellulase enzymes is in general agreement with the observed level of processivity and suggests that this functional-kinetic property is manifest in a relatively limited range of linker lengths, which appear to be in co-evolutionary link with the particular step size along their typical substrate. This has been also suggested by the behavior of the related mechanochemical motors kinesin-1 and kinesin-2, the degree of processivity of which sharply changes by changing the length of their linker regions [15
]. This feature is also underlined by the observation that short and long linkers are entirely missing in DLD-type processive enzymes.
In a broader functional context, we suggest that this observed behavior is a special case of the entropic chain functions of IDPs/IDRs and appears as a conceptual extension of mechanisms, such as fly casting [27
] and monkey-bar mechanism [28
]. Processivity appears to draw on all these mechanisms and may represent one of the primary benefits of the flexibility emanating from structural disorder [25
]. This type of function cannot be supported by a structured protein; thus it is an appealing addition to the functional arsenal of structural disorder, understanding of which may even enable the design and generation of enzymes of improved capacity for the needs of biotechnology.