 Research article
 Open Access
 Published:
Investigation of sequence features of hingebending regions in proteins with domain movements using kernel logistic regression
BMC Bioinformatics volume 21, Article number: 137 (2020)
Abstract
Background
Hingebending movements in proteins comprising two or more domains form a large class of functional movements. Hingebending regions demarcate protein domains and collectively control the domain movement. Consequently, the ability to recognise sequence features of hingebending regions and to be able to predict them from sequence alone would benefit various areas of protein research. For example, an understanding of how the sequence features of these regions relate to dynamic properties in multidomain proteins would aid in the rational design of linkers in therapeutic fusion proteins.
Results
The DynDom database of protein domain movements comprises sequences annotated to indicate whether the amino acid residue is located within a hingebending region or within an intradomain region. Using statistical methods and Kernel Logistic Regression (KLR) models, this data was used to determine sequence features that favour or disfavour hingebending regions. This is a difficult classification problem as the number of negative cases (intradomain residues) is much larger than the number of positive cases (hinge residues). The statistical methods and the KLR models both show that cysteine has the lowest propensity for hingebending regions and proline has the highest, even though it is the most rigid amino acid. As hingebending regions have been previously shown to occur frequently at the terminal regions of the secondary structures, the propensity for proline at these regions is likely due to its tendency to break secondary structures. The KLR models also indicate that isoleucine may act as a domaincapping residue. We have found that a quadratic KLR model outperforms a linear KLR model and that improvement in performance occurs up to very long window lengths (eighty residues) indicating longrange correlations.
Conclusion
In contrast to the only other approach that focused solely on interdomain hingebending regions, the method provides a modest and statistically significant improvement over a random classifier. An explanation of the KLR results is that in the prediction of hingebending regions a longrange correlation is at play between a small number amino acids that either favour or disfavour hingebending regions. The resulting sequencebased prediction tool, HingeSeek, is available to run through a webserver at hingeseek.cmp.uea.ac.uk.
Background
Protein domains have various definitions within Biochemistry [1]. From a structural perspective a domain is characterised as a globular, spatially separate part of a protein and methods have been developed to recognise them from this property [2]. They are considered to be able to fold independently of other parts of the protein and are associated with a distinct function. This lends them the ability to act as a fundamental component of evolutionary change. For protein structure databases such as SCOP [3], SCOP2 [4] and CATH [5] they form the basic element of classification. They can be identified from sequence homology using methods such as Pfam [6] where multiplesequence alignments of family members of a domain are encoded as hidden Markov models.
It is now an established fact that conformational change is integral to protein function [7, 8]. A common class of movement is a domain movement in proteins comprising more than one domain [9,10,11,12]. Several methods have been developed to identify domains from the movement itself [13,14,15,16,17,18] and in this context they have been called “dynamic domains”. The relative movement of dynamic domains is controlled by socalled hingebending regions located between the domains. These normally comparatively short regions collectively control the domain movement [10] as has been demonstrated using inversekinematics Monte Carlo in glutamine binding protein where the known domain movement was reproduced almost perfectly when only 11 of the 226 residues situated at the two hingebending regions were allowed to flex [19]. In an early application of the DynDom method it was found that hingebending regions are often situated at the termini of βsheets and αhelices [10].
To date very little work has been carried out to determine whether hingesite features are reflected in the sequence. Flores et al. [20] annotated hingebending regions from the Database of Macromolecular Motion (DBMM) [21] to form their “Hinge Atlas” dataset and performed statistical analyses to create a predictor for hinge sites from sequence alone. Hinge sites were identified using the FlexProt program [22]. They calculated logodds frequencies scores for a 17residuelong sliding window, assigning the central residue to a hingebending region if the resulting accumulated score was above a threshold. The results achieved did not appear to be significantly different to a random assignment. They incorporated information about secondary structure and active site location into the predictor, “HingeSeq”, which improved predictive power. They did not quote the area under the ROC curve (AUROC) but we estimated it from their figure to be approximately 0.65.
Kuznetsov [23] reports using support vector machines (SVM) to predict “conformational switches” from sequence, which were described as areas of flexibility that drive conformational change. The basic data used also came from the DBMM but the sites identified, based on changes in mainchain dihedral angles, were not exclusively located at hingebending regions. Using a window length of 11 residues, an AUROC of 0.64 was found, which increased to 0.69 when profiles were used. The method has been implemented at the webserver FlexPred [24]. Bodén and Bailey [25] presented a method, also based on the DBMM, which predicted “conformational variability” based on secondary structure prediction uncertainty for which a neural network was used. A window length of 15 was used and an AUROC of 0.64 was reported.
This work relates also to the study of linker regions; polypeptide regions that link two domains [26, 27]. The difference between these linker region studies and hingebending region/conformationalswitch region studies, is that the latter were identified from conformational change, whereas the former were identified purely on structural features. There is an increasing interest in the dynamic properties of linker regions as their rational design would benefit the efficacy of therapeutic fusion proteins constructed using recombinant DNA technology [28].
A feature of the DynDom program is that it determines not only dynamic domains but also hingebending regions, as can be seen in the example of glutamine binding protein in Fig. 1. Dynamic domains are determined based on their rotational properties and hingebending regions are those regions within which a rotational transition occurs in going from one dynamic domain to another. This connects directly with what “bending” really means. The exact method for assigning bending regions is described in detail by Hayward and Lee [29]. This precise definition of a bending region lends itself to the aim of this study. Here we trained a range of Kernel Logistic Regression (KLR) models on protein sequences with hingesite annotation from examples that showed a clear hingebending movement in the two main DynDom databases in order to understand sequence properties of hingebending regions and to produce a hinge site predictor from sequence.
Results
Hinge statistics
The Hinge Index, HI(a), for each amino acid, a, is shown in Fig. 2 for all three Group 1 datasets, that is Group1_90%, Group1_40% and Group1_20%. A negative HI(a) would indicate an amino acid that is unfavourable to hinge regions, a value of zero, an amino acid that has no preference, and a positive value an amino acid favourable to hinge regions. Although the results are generally supportive of those found by Flores et al., they are statistically significant only for a few amino acids in both studies. For Flores et al. Ser and Gly had the highest significant HI values. Here, Pro has the highest significant HI value at all three levels of filtering. We also found Ser to have a high significant HI value at 90 and 40% filtering, but contrary to expectation, Gly was not in the top four at any level of filtering.
At all levels of filtering, Cys received the most negative significant HI value and by a large margin. Phe and Met also disfavour hinge regions, Phe being the amino acid with the most negative HI value for Flores et al.. The βbranched amino acids Ile, Val and Thr all seem to weakly disfavour hinge regions although the results are not statistically significant.
The equivalent analysis on the Group2_90% is shown in Additional Figure 1. The results broadly agree with the Group1_90% results.
KLR on 90% sequence identity set
Group 1
We trained KLR models with linear, quadratic, cubic, and RBF kernels on the training subset from Group1_90% (see Table 1). Each KLR model was constructed across a range of window lengths, w = [1, 101], and tested on the test set comprising 10% of the whole set selected at random. ROC curves were created for each window length and each kernel, plotting the rate of true positive outcomes against the rate of false positive outcomes. The AUROCs were calculated, giving a measure of performance for each combination of window length and kernel, as a number between zero and one, where higher numbers represent better performance. Figure 3a shows how these AUROCs change across window lengths for each kernel in Group1_90%. A classifier with an AUROC of 0.5 would be equivalent to assigning samples to the “hingebending region” or “not hingebending region” classes at random. There are two main things to notice about these results. First is that there is improvement in AUROC up until very long window lengths. This result is in contrast to previous studies on hingebending/conformationallyvariable regions where windows of length less than 25 residues were used by Kuznetsov [23], a window of 17 residues by Flores et al. [20], and a window of 15 residues by Bodén and Bailey [25]. Here we see an improvement in AUROC with window lengths up to 80–90 residues. This suggests that if the window spans from one hingebending region to the next it can help prediction. The other noticeable feature is that the quadratic, cubic, and RBF kernels all seem to outperform the linear approach. Additional Table 1 shows a matrix of pvalues for the pairwise comparisons of the AUROC for the four different models for window length 99 residues using Sun and Xu’s implementation [30] of the method by DeLong et al. [31]. The DeLong et al. method tests the null hypothesis that the difference in the empirical AUROCs can be adequately explained by the variance of the estimator. The null hypothesis is rejected when p < 0.05. This shows that all nonlinear models significantly outperform the linear model, but that the nonlinear models do not all significantly outperform each other. That the cubic model and RBF models do not improve performance over the quadratic model suggests that the quadratic terms are mainly where the improvement lies. This implies that there exists a correlation between certain pairs of residues at different positions within the window. The maximum value for the AUROC of 0.75 occurred for the quadratic model with a window length of 87 residues. The maximum value of the AUROC for the linear model was 0.69 with a window length of 99 residues.
As stated in the Methods section, the ratio of positive to negative cases was adjusted to 1:9 for the training set, but in the test set the proportion of residues that are in hinge regions is only 0.0294 indicating a large class imbalance. In Additional Figure 2(A) we show a set of ROC curves and their AUROCs from the quadratic model with a window length 81 that uses different proportions of positive to negative cases in the training sets. We also show in Additional Figure 2(B), plots of how the AUROC varies with this proportion for different window lengths. These results confirm that KLR is reasonably robust to class imbalance as there is little change in the AUROCs with change in this proportion.
In Additional Figure 3 we show the PrecisionRecall plot for window length 81. Such a plot emphasises the classification of positive examples. The area under the PrecisionRecall plot (AUPRC), which is dependent on the class imbalance ratio, is 0.1785. A random classifier would give an AUPRC of 0.0294, the proportion of hinge residues in the test set. Additional Figure 4 shows the AUPRCs plotted against window length for the four different KLR models. The result mirrors the equivalent plot for the AUROCs.
Group 2
The Group2_90% was used for the same set of experiments as Group1_90%, although due to the greatly increased computational expense resulting from the use of this larger training set, fewer window lengths were tried although they spanned the same range (Fig. 3b). Again we found the same increase in performance with window length and the same improvement of the nonlinear models over the linear model. The matrix of pvalues in Additional Table 2 determined with DeLong et al.’s method, shows that the difference between the nonlinear models and the linear model was statistically significant. In comparison with Group1_90%, each model performed worse at most window lengths indicating the negative influence of the less strict selection criteria for Group2_90%.
KLR on 40% sequence identity set
We considered whether the 90% sequence identity might permit similar sequences to be present in both training and test sets. The Group1 dataset contains 48 chains from immunoglobulins; pairwise comparisons between these sequences resulting in sequence identities ranging between 19.2 and 88.9%. We repeated the experiment for linear and quadratic models on the Group1_40% dataset, within which pairs of structures are less likely to be homologous [32]. This reduced the number of immunoglobulins included to 3 of 171 proteins. As this reduced the size of the dataset (see Table 1), we performed 10fold cross validation (nested crossvalidation was used in order to obtain an unbiased performance estimate [33]). Figure 4a shows the mean AUROC of the folds across windows of length 3 to 41 in increments of 2, and 41 to 101 in increments of 10. The results for both linear and quadratic kernels were poorer than the Group1_90% results, which is expected as there is less data in the training set. The models both improved at longer window lengths: the mean AUROC for the quadratic kernel was 0.61 achieved at window length 81, and the linear kernel peaked at a mean AUROC of 0.57 at 61 residues. pvalues for paired ttests across the folds for different window lengths is shown in Additional Figure 5. Additional Figure 5 shows that the longer the window, the lower the pvalue becomes for the difference between the quadratic and linear model. At a window length 81 the pvalue is 0.004 indicating a statistically significant improvement of the quadratic model over the linear model at long window lengths. Across the folds the AUPRC has a value mean value of 0.0415 compared to a mean ratio of hinge residues to all residues of 0.0232.
KLR on 20% sequence identity set
We repeated these experiments using the Group1_20% dataset. As our original dataset is relatively small, filtering at the 20% level reduces the amount of data to an even lower level (see Table 1). Again we performed 10fold cross validation. Figure 4b shows the mean AUROC of the folds across the same range of window lengths used for 40 and 90% filtering. As expected the results for both linear and quadratic kernels were poorer than the 90 and 40% results. Although the difference between the linear and quadratic models was not found to be significant using the paired ttest (which is likely due to the small amount of data), we do see the same trend as seen for the 90 and 40% results; that is an improvement in the AUROC of the quadratic model over the linear model at longer window lengths.
Across the folds the AUPRC has a value mean value of 0.0390 compared to a mean ratio of hinge residues to all residues of 0.0213.
Analysis of model weights
In this section, we analyse the weights from the quadratic and linear kernels, at their optimal window lengths: 87 for Group1_90%, 81 for Group1_40%, and 87 for Group1_20%. The primal weight vector can be computed for finite feature spaces such as that of the linear and quadratic kernels, using Eq. 8.
Linear terms
Figure 5 shows example plots of the linear weight distribution for given amino acids across the window. The scale of the weights differed between the linear and quadratic models, so each weight is represented as a proportion of the strongest weight applied by the model to the amino acid.
While there is some disagreement between the models, strong peaks and troughs can be observed at the same window positions for all three models. Pro was associated with strong positive weights in and around the central position, with negative weights ±40 residues from the central position. Pro has the highest positive weight of any amino acid at the central window position confirming the Hinge Index result. The weights in the Cys plots are mostly negative. It has the lowest valued weights at the central window position out of all amino acids. Interestingly it has pronounced positive weights around ±20 residues from the central position. The weights in the Ile plot fluctuate but all three models show strong positive weights around 5 residues on the Nterminal side of the central position and a smaller peak 5 residues after. These charts are not all approximately symmetrical; the Trp plot shows a strong positive peak around the end of the window, with no corresponding peak at the start.
Product terms
The feature space for the quadratic kernel includes features corresponding to the pairwise products of the original input attributes. The weights associated with product terms in the feature vectors give an indication of the strength of the importance of pairs of residues at different positions within the sliding window. These can be visualised for each amino acid pair by plotting them as a heat map, where each axis represents a position within the sliding window at which a residue occurs.
The heat map in Fig. 6 shows the weights associated with combinations of Cys and Pro residues according to the quadratic model trained for the Group1_40% dataset. A patch of positive weights at position (20–25, 0–10) may indicate that such a combination is favoured. Structurally this would suggest a pair of domains with Pro located at a hingebending region and Cys located at an intradomain region on the Cterminal side. At this current time we cannot rule out the possibility that these correlations are an artefact of the small sample we have of nonhomologous proteins with clear domain movements.
As optimal AUROCs predominantly occurred at window lengths of either 81 or 87, we include in Additional Table 3, AUROCs at both these window lengths (although AUROCs are not available for window length 87 on Group1_40% as we did not perform computations at this window length). The results show there is little or no difference between the AUROCs at these two window lengths.
HingeSeek web server
We have produced a tool, called “HingeSeek”, which is available to run from a web server at hingeseek.cmp.uea.ac.uk. The server offers sequenceonly hinge predictions, converting input sequences into windowed oneofn encoded feature vectors and classifying each residue as hinge or nonhinge based on a selected threshold. The sequence is then coloured according to the classification, and labelled with the confidence level.
HingeSeek was created by bootstrapping the training data from Group1_90%. One hundred models were trained using the quadratic KLR model with the optimal window length of 87. Data was sampled with replacement creating training sets the same size as the original Group1_90% set. To allow unbiased assessment of the model’s predictions, there is a sequence identity threshold parameter. When a sequence is entered by the user, an ensemble is created such that no members of the ensemble were trained on any sequences having a greater sequence identity than the threshold with the input sequence. The weights are extracted from the selected models and averaged to create an aggregated model. This enables the tool to be used as a fair benchmark for comparison with competing approaches. In addition to allowing users to predict hingebending regions, the web server also includes an interactive weight explorer, which allows users to investigate the weights that the model assigned to amino acid pairings, by dynamically generating charts like Fig. 6.
Discussion
We trained a range of KLR models on sequences taken from the DynDom database in order to understand sequence features of hingebending regions and to predict their locations from sequence alone.
With Group1_90%, a maximum AUROC of 0.75 was achieved. This contrasts favourably with Flores et al. [20] who could not achieve any predictive value using just Hinge Index information using the DBMM dataset also filtered at 90% sequence identity. With Group 1_40% and Group1_20%, the AUROC of the best KLR model was lower than the AUROC for the best KLR model using Group1_90%, probably due to the small amount of data available at these levels of sequence identity.
Beyond producing a sequencebased predictor for hinge regions, this work provides insight into what kinds of residue favour or disfavour hinge regions and hints at possible relationships between them. Broadly the residues found to favour hinge sites are those with small side chains confirming the finding by Flores et al. [20]. Ser strongly favours a hinge site even more so than Gly which, in contrast to Flores et al., we find only weakly favours hinge regions. Both for Group 1 and Group2, the Hinge Index analysis shows that Pro is the most favourable residue to be located at a hinge region and Cys the least favourable. This result is supported by an analysis of the weights of the linearterms in the KLR models. The fact that Pro favours hingebending regions is unexpected as in contrast to all other amino acids rotation about its ϕ dihedral is severely restricted which one would think would inhibit its ability to act as a hingebending residue. This result concurs with studies on linker regions [26, 27] identified on structural features only. Such regions were intentionally omitted from our datasets as positive cases in order to be certain that those included were confined to those that demonstrably facilitate hinge bending. We believe the reason for Pro being located in these regions is that it often acts as a terminator for secondary structure elements and therefore appears at hinge regions because they are also often located at the terminal regions of secondary structures [10]. Cys is highly disfavoured at bending regions which can be explained by the fact that many Cys residues form disulphide bonds helping to rigidify the local backbone. Positive weights for Cys at the ± ~ 20 positions probably indicate the role it plays in stabilising a domain via crosslinking. Interestingly Ile appears to act as a domaincapping residue. The preference of some residues to be situated in bending regions and the preference of others for being located within a globular domain may explain why we see improvement in prediction up to comparatively long window lengths.
The consistently better performance of the quadratic kernel over the linear kernel at very long window lengths implies a correlation between amino acid locations which we believe occurs between a small number of amino acids, such as Pro and Cys, that particularly favour or disfavour hinge bending regions.
Conclusions
We have used statistical methods and machine learning methods to investigate sequence features of hingebending regions. This work presents an example of an attempt to analyse sequence features involved in the structuredynamic relationship. There is an increased interest in these regions particularly in their role as linkers in therapeutic fusion proteins. First, we revisited the Hinge Index measure introduced by Flores et al. [20]. The results broadly confirm their findings for the propensities of particular amino acids to occur in hingebending regions. However, there are some differences, most notably the finding that proline is the amino acid that has the highest propensity to occur in a hingebending region. This is thought to be due to its secondarystructure breaking tendency as it is at the termini of secondary structures that hinge bending often occurs. Flores et al. found that the Hinge Index alone could not be used to produce a reliable predictor and so here we have used KLR. Although we have produced a tool with useful predictive power it has not achieved the same level of predictive power as when machine learning methods are applied to secondary structure prediction from sequence [34]. This problem represents a case where there is a large class imbalance with the number of intradomain residues vastly outweighing the number of hingebending residues. This means that with a limited amount of data, and as our results indicated, only a few of the 20 amino acids having expressed any strong preference for or aversion of hinge regions, the number of false positives is likely to be high. Using KLR models of increasing complexity we have found an interesting and quite unusual feature for the prediction of hingebending regions, namely that the quadratic model outperforms the linear model particularly at very long window lengths (in comparison to other methods that have been applied to the prediction of hingebending/conformationallyvariable regions). This result points to prediction performance being enhanced by the correlation between those residues that strongly favour or disfavour hingebending regions at a considerable distances apart along the chain. Understanding the role that particular amino acids play in the formation of hinge regions will be of interest to those who practise protein engineering, particularly those who design linker regions in therapeutic fusion proteins.
Methods
Dataset
The primary data comprised 5248 domain movements from unique pairs of structures analysed by the DynDom program. These are deposited in both the usercreated database [35] and the nonredundant database [36]. We selected only those that were uncontestably clear domain movements based on filtering criteria. We created two datasets, “Group 1” a strictly filtered group, and “Group 2” filtered based on more permissive criteria. Table 1 shows the filtering criteria for these two groups. We take the sequence of the Conformer 1 structure (the two structures submitted are assigned as “Conformer 1” and “Conformer 2” at the DynDom webserver by the expert user) with the residues annotated as hingebending or intradomain. Figure 1 shows glutamine binding protein, a typical member of Group 1. In the usercreated set there is a great deal of redundancy. We follow Flores et al. [20] initially by filtering at 90% sequence identity on each group to ensure that no two sequences are selected for the same group if they have a sequence identity of 90% or higher. To achieve this we used the program CDHit [37]. The total counts for the data sets were 241 sequences in Group 1 and 372 sequences in Group 2. Group 1 can be regarded as containing clear hinge regions whereas Group 2 may contain some less hingelike regions. Lists of the PDB structures in Groups 1 and 2 at 90% filtering are given in the Additional Data 1. These pairs identify the domain movement which can be viewed at the DynDom website.
We also filtered the datasets at 40 and 20% sequence identity thresholds using CDHit to assess the effect of removing homologous proteins. In the Results section we refer to the different datasets as Group1_90%, Group2_90%, Group1_40% and Group1_20%.
Hinge Index
Flores et al. [20] proposed the Hinge Index, HI(a), for a given amino acid, a, as:
which, is the loglikelihood ratio for the occurrence of amino acid a in a hinge region to its occurrence in the population as a whole. It is a measure of the propensity of an amino acid for a hinge region. p(a) is the probability of amino acid a irrespective of region and p(a h) is the probability of amino acid a given it is in a hinge region, h. These probabilities were estimated from frequencies calculated using the annotated sequence data. Significance testing of HI(a) is performed using the hypergeometric distribution as outlined in detail by Flores et al. pages 6–7. The null hypothesis is that the observed number of occurrences of an amino acid of a particular type in hinge regions is the result of the random assignment of that amino acid to hinge regions according to its probability of occurrence in any region derived from its overall frequency. The alternative hypothesis is that it is not a random assignment with probabilities derived from their overall frequencies. Following Flores et al., the null hypothesis is rejected when p < 0.05.
Kernel logistic regression
To build the training and test data sets from the sequence and bending region data, a sliding window of length w residues was placed over each sequence, resulting in subsequences of length w residues. If w is odd then the central residue of the window can either be in an intradomain region or a hingebending region. To get from our windowed sequence to a suitable input vector we employ “oneofnencoding”. For each window i the sequence is encoded as a 24w component input vector, x_{i}, where for each position in the window, 24 rows are assigned, each of which corresponds to the one of 24 “characters” in our alphabet: one character for each of the 20 standard amino acids plus “B”, “X” and “Z”, standing for ambiguous amino acids and “” as a dummy character for those positions in the window that are beyond a terminus. The value of each of the 24 rows is set to 0 for each residue apart from the row of the residue at the corresponding window position which is set to 1.
Those windows with the central residue in an intradomain region were negatively labelled and have a target value for KLR of t_{i} = 0, and those with the central residue in a hingebending region were positively labelled and given a target value of t_{i} = 1. The number of negatively labelled records in the training set greatly outnumbered the number of positively labelled records, so this ratio in the training set was altered by randomly discarding negatively labelled examples. We elected to use a 1:9 proportion for the positive to negative cases for all training sets. In the Results section we show that variation of the proportion of positive to negative cases in the training set did not affect the AUROC.
KLR was applied to the data using UEA’s MATLAB Generalized Kernel Machine toolbox [38]. KLR [39] constructs a model of the form:
where b is a scalar bias parameter, w is a vector of primal model parameters, and ϕ(x) is the representation of x in a fixed feature space. The logit link function constrains the output of the model to lie between zero and one. Viewing this output as an aposteriori probability of belonging to the “hinge” class, we classify test residues as part of a hingebending region if the output is above a certain threshold, and part of an intradomain region if the output is below the threshold.
Rather than define the nonlinear transformation, ϕ(x), directly, it is implicitly defined by a kernel function, \( \mathcal{K} \), giving the inner product between vectors in the feature space,
where x and x^{′} are arbitrary vectors in the input space. A valid kernel function is one that obeys Mercer’s conditions; i.e. the resulting kernel matrix, K, is positive semidefinite for any set of points in the input space. We used three kernels starting with the linear kernel function, a straightforward scalar product of the input vectors:
The polynomial kernel of Eq. 5, maps the input vector into a higher dimensional feature space where new features are created from all monomials of order d or less of the original features. This allows nonlinear separations of the data without requiring an enumeration of the possible combinations.
In this study, the kernel parameter d was set at two (for a quadratic kernel) or three (for a cubic kernel), and c is a hyperparameter. The final kernel function used was the radial basis function (RBF) kernel:
where θ is a hyperparameter controlling the sensitivity of the kernel.
Assume we are given a training set of ℓ examples, where x_{i} represents an input vector and t_{i} and y_{i} are, respectively, the expected and predicted outcome for the i^{th} training example. The optimal values of the primal model parameters, w, and bias, b, are found using the iteratively reweighted least squares training procedure [40] to minimise a regularised “crossentropy” cost function:
This optimisation problem is more conveniently solved in the dual representation, where the primal parameters are expressed in terms of the dual parameters:
where α is vector of dual model parameters. From Eq. 2, Eq. 3 and Eq. 8, the equation used to calculate an expected outcome from an input vector is:
The regularization parameter, γ, in Eq. 7 along with other hyperparameters such as the kernel parameter θ in Eq. 6 and the polynomial kernel’s hyperparameter c in Eq. 5, are tuned using the NelderMead simplex algorithm [41] to minimise an approximate leaveoneout crossvalidation estimate of the crossentropy loss [40], which can be computed efficiently as a byproduct of the training procedure, i.e. the leaveoneout crossvalidation is performed on the training set.
Availability of data and materials
All the data used are available from the Protein Data Bank (PDB) – see Additional Data 1 for list of accession codes – at wwpdb.org and from the DynDom website at www.cmp.uea.ac.uk/dyndom.
Abbreviations
 PDB:

Protein Data Bank
 KLR:

Kernel logistic regression
 DBMM:

Database of macromolecular motion
 HI:

Hinge Index
 ROC:

Receiver operator characteristic
 AUROC:

Area under ROC curve
 AUPRC:

Area under precisionrecall curve
References
Ponting CP, Russell RR. The natural history of protein domains. Annu Rev Biophys Biomol Struct. 2002;31:45–71.
Wernisch L, Wodak SJ. Identifying structural domains in proteins. In: Bourne PE, Weissig H, editors. Structural bioinformatics: WileyLiss; 2003.
Murzin AG, Brenner SE, Hubbard T, Chothia C. SCOP  a structural classification of proteins database for the investigation of sequences and structures. J Mol Biol. 1995;247(4):536–40.
Andreeva A, Howorth D, Chothia C, Kulesha E, Muzin AG. SCOP2 prototype: a new approach to protein structure mining (vol 42, pg D310, 2014). Nucleic Acids Res. 2014;42(18):11847.
Orengo CA, Michie AD, Jones S, Jones DT, Swindells MB, Thornton JM. CATH  a hierarchic classification of protein domain structures. Structure. 1997;5(8):1093–108.
ElGebali S, Mistry J, Bateman A, Eddy SR, Luciani A, Potter SC, et al. The Pfam protein families database in 2019. Nucleic Acids Res. 2019;47(D1):D427–D32.
Hammes GG. Multiple conformational changes in enzyme catalysis. Biochemistry. 2002;41(26):8221–8.
Teague SJ. Implications of protein flexibility for drug discovery. Natl Rev. 2003;527:527–41.
Gerstein M, Lesk AM, Chothia C. Structural mechanisms for domain movements in proteins. Biochemistry. 1994;33(2):6739–49.
Hayward S. Structural principles governing domain motions in proteins. Proteins. 1999;36:425–35.
Lesk AM, Chothia C. Mechanisms of domain closure in proteins. J Mol Biol. 1984;174:175–91.
Schulz GE. Domain motions in proteins. Curr Opin Struct Biol. 1991;1:883–8.
Hayward S, Berendsen HJC. Systematic analysis of domain motions in proteins from conformational change: new results on citrate synthase and T4 lysozyme. Proteins. 1998;30:144–54.
Hayward S, Kitao A, Berendsen HJC. Model free methods to analyze domain motions in proteins from simulation. A comparison of a normal mode analysis and a molecular dynamics simulation of lysozyme. Proteins. 1997;27:425–37.
Hinsen K, Thomas A, Field MJ. Analysis of domain motions in large proteins. Proteins. 1999;34:369–82.
Wriggers W, Schulten K. Protein domain movements: detection of rigid domains and visualization of hinges in comparisons of atomic coordinates. Proteins. 1997;29:1–14.
Poornam GP, Matsumoto A, Ishida H, Hayward S. A method for the analysis of domain movements in large biomolecular complexes. Proteins. 2009;76(1):201–12.
Veevers R, Hayward S. Methodological improvements for the analysis of domain movements in large biomolecular complexes. Biophys Physicobiol. 2019;16:328–36.
Hayward S, Kitao A. Monte Carlo sampling with linear inverse kinematics for simulation of protein flexible regions. J Chem Theory Comput. 2015;11(8):3895–905.
Flores SC, Lu LJ, Yang JL, Carriero N, Gerstein MB. Hinge Atlas: relating protein sequence to sites of structural flexibility. BMC Bioinformatics. 2007;8:167.
Gerstein M, Krebs W. A database of macromolecular motions. Nucleic Acids Res. 1998;26(18):4280–90.
Shatsky M, Nussinov R, Wolfson HJ. Flexible protein alignment and hinge detection. Proteins. 2002;48(2):242–56.
Kuznetsov IB. Ordered conformational change in the protein backbone: prediction of conformationally variable positions from sequence and lowresolution structural data. Proteins. 2008;72(1):74–87.
Kuznetsov IB, McDuffle M. FlexPred: a webserver for predicting residue positions involved in conformational switches in proteins. Bioinformatian. 2008;3(3):134–6.
Boden M, Bailey TL. Identifying sequence regions undergoing conformational change via predicted continuum secondary structure. Bioinformatics. 2006;22(15):1809–14.
Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol. 1990;211(4):943–58.
George RA, Heringa J. An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng. 2002;15(11):871–9.
Chen XY, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013;65(10):1357–69.
Hayward S, Lee RA. Improvements in the analysis of domain motions in proteins from conformational change: DynDom version 1.50. J Mol Graph Model. 2002;21(3):181–3.
Sun X, Xu WC. Fast implementation of Delong’s algorithm for comparing the areas under correlated receiver operating characteristic curves. IEEE Signal Process Lett. 2014;21(11):1389–93.
DeLong ER, DeLong DM, ClarkePearson DL. Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. Biometrics. 1988;44(3):837–45.
Sander C, Schneider R. Database of homologyderived protein structures and the structural meaning of sequence alignment. Proteins. 1991;9(1):56–68.
Cawley GC, Talbot NLC. On overfitting in model selection and subsequent selection bias in performance evaluation. J Mach Learn Res. 2010;11:2079–107.
Rost B. Review: protein secondary structure prediction continues to rise. J Struct Biol. 2001;134(2–3):204–18.
Lee RA, Razaz M, Hayward S. The DynDom database of protein domain motions. Bioinformatics. 2003;19(10):1290–1.
Qi G, Lee RA, Hayward S. A comprehensive and nonredundant database of protein domain movements. Bioinformatics. 2005;21(12):2832–8.
Li WZ, Godzik A. CDhit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics. 2006;22(13):1658–9.
Cawley GC, Janacek GJ, Talbot NLC. Generalised kernel machines. 2007 International Joint Conference on Neural Networks. 2007.
Zhu J, Hastie T. Kernel logistic regression and the import vector machine. Advances in neural information processing systems. 2002.
Cawley GC, Talbot NLC. Efficient approximate leaveoneout crossvalidation for kernel logistic regression. Mach Learn. 2008;71(2–3):243–64.
Nelder JA, Mead R. A simplexmethod for function minimization. Comput J. 1965;7(4):308–13.
Acknowledgements
We thank Professor James MilnerWhite for helpful discussions.
Funding
RV was funded by a University of East Anglia studentship.
Author information
Authors and Affiliations
Contributions
RV, GC and SH contributed to the design of the approach. RV did the computations and designed and implemented the HingeSeek webserver. RV, GC and SH helped to write the manuscript. RV, GC and SH read and approved the final manuscript.
Corresponding authors
Ethics declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Additional file 1.
Data formatted list of PDB accession codes and chain IDs of pairs of structures used in Groups 1 and 2.
Additional file 2: Table S1.
Table giving matrix of pvalues for the pairwise comparisons of the AUROC for the linear, quadratic, cubic and RBF models for Group1_90% dataset.
Additional file 3: Table S2.
Table giving matrix of pvalues for the pairwise comparisons of the AUROC for the linear, quadratic and cubic models for Group2_90% dataset.
Additional file 4: Table S3.
Table for comparison of AUROCs for window lengths 81 and 87.
Additional file 5: Figure S1.
HingeIndex values for amino acids evaluated from Group2_90% dataset.
Additional file 6: Figure S2.
(A) ROC curves for the quadratic model with window length 81 on Group1_90% with various proportions of positive to negative training examples. (B) Plots of the AUROC against proportion of positive to negative training examples for different window lengths.
Additional file 7: Figure S3.
PrecisionRecall curve for Group1_90%.
Additional file 8: Figure S4.
Area under PrecisionRecall curves for different KLR models at different window lengths for Group1_90% dataset.
Additional file 9: Figure S5.
pvalues at different window lengths for the Group1_40% dataset determined by doing a paired ttest of the AUROC between the linear and quadratic KLR models.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
About this article
Cite this article
Veevers, R., Cawley, G. & Hayward, S. Investigation of sequence features of hingebending regions in proteins with domain movements using kernel logistic regression. BMC Bioinformatics 21, 137 (2020). https://doi.org/10.1186/s1285902034643
Received:
Accepted:
Published:
DOI: https://doi.org/10.1186/s1285902034643
Keywords
 Protein conformational change
 Domain closure
 Hinge axis
 Linker region